Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis, an often fatal disease in tropical countries. Burkholderia thailandensis is a non-virulent but closely related species. Both species are soil saprophytes but are almost never isolated together.B. pseudomallei affects the growth of B. thailandensis. First, we found that six different isolates of B. pseudomallei inhibited the growth of B. thailandensis on LB agar plates. Second, our results indicated that 55% of isolated strains of B. pseudomallei produced a secreted compound that inhibited the motility but not the viability of B. thailandensis. Analysis showed that the active compound was a pH-sensitive and heat-labile compound, likely a protein, which may affect flagella processing or facilitate their degradation. Analysis of bacterial sequence types (STs) demonstrated an association between this and motility inhibition. The active compound was produced from B. pseudomallei during the stationary growth phase.We identified two mechanisms by which B. pseudomallei inhibits both the growth and motility of its close relative B. thailandensis. The latter phenomenon appears to occur via a previously unreported mechanism involving flagellar processing or degradation.Taken together, our results indicate that The online version of this article (doi:10.1186/s12866-015-0395-7) contains supplementary material, which is available to authorized users. B. pseudomallei is a Gram-negative bacillus and the cause of melioidosis. Most cases of infection are reported from northeast Thailand and northern Australia, although an increasing number of cases are being reported from across Southeast Asia, and from Africa and south America [B. pseudomallei is an environmental saprophyte, and infection arises following bacterial inoculation, inhalation or ingestion [ America . B. pseungestion . The ranngestion . In Thaingestion , and theB. pseudomallei have been performed to address a range of questions, including mapping of geographic distribution, the effect of sampling depth and season on positivity of B. pseudomallei in soil and water, and the phylogeny of environmental isolates [2) in northeast Thailand was the genetic diversity of B. pseudomallei within and between sampling points. Genotyping of 600 primary culture plate colonies from 3 sampling points showed that each contained three or four different B. pseudomallei clones, with little overlap in genotype between samples but a predominance of a single genotype within a given sample [B. pseudomallei lineages and/or other microbial species or genera.Environmental surveys to detect isolates . A strikn sample . One expB. pseudomallei is genetically closely related to the non-pathogenic B. thailandensis [B. pseudomallei. Although B. thailandensis is rarely searched for systematically during environmental surveys of B. pseudomallei, B. thailandensis grows on the same culture media and the colony morphology on agar can be difficult to distinguish from B. pseudomallei. As a result, B. thailandensis is frequently isolated during soil sampling and discarded after identification. A review of environmental study indicates that B. pseudomallei and B. thailandensis are rarely isolated from the same sampling location [B. pseudomallei to B. thailandensis was 1.7, 0.9, 0.5 and 0.4 in northeastern, southern, northern and central regions, respectively [B. pseudomallei and B. thailandensis in the northeast was associated with a higher prevalence of melioidosis in this region compared with others. One explanation for this observation is competition between B. pseudomallei and B. thailandensis.andensis ,6, whichlocation . A studyB. thailandensis over other environmental bacterial species through a range of mechanisms. B. thailandensis secretes an antibiotic that inhibits the growth of Bacillus subtilis [B. thailandensis more susceptible to cell contact-induced stasis by Pseudomonas putida, Pseudomonas fluorescens and Serratia proteamaculans. B. thailandensis lacking T6SS-1 is also rapidly displaced from mixed biofilms with P. putida, whereas wild-type persists and overgrows the competitor [B. pseudomallei and B. thailandensis have also been reported to have a contact-dependent growth inhibition (CDI) system mediated by the CdiB/CdiA family of two-partner secretion proteins [Several studies have provided evidence for a competitive advantage of subtilis . Inactivmpetitor . B. pseuproteins ,11.B. pseudomallei and B. thailandensis isolates associated with human infection  and the environment (both species), and to explore the relationship between inhibition and genotype.The aim of this study was to use in vitro assays to investigate competition (including viability and motility) between a range of B. pseudomallei and B. thailandensis, the individual growth of six B. pseudomallei  and B. thailandensis Bt6 were compared in LB broth. Using a starting inoculum of 1 × 105 cfu/ml, log and stationary phase occurred at 2 h and 12 h, respectively, for all isolates. There was no difference in doubling time between B. pseudomallei and B. thailandensis. The average doubling time for B. pseudomallei K96243, H1244a, 1106b, 1026b, 1710a and B4 were 38.7, 38.3, 38.4, 38.4, 38.6 and 38.5, and for B. thailandensis Bt6 was 39.8 min, respectively.Different growth rates may affect the number of viable bacteria in the growth inhibition assay. Thus, prior to observation of competition between B. pseudomallei 1710a inhibited B. thailandensis Bt6 when mixed at ratios from 1000:1 to 10:1  and one soil B. pseudomallei isolate (B4) against B. thailandensis Bt6 at a ratio of 1000:1. This demonstrated a 1 to 2 log increase for six different B. pseudomallei isolates and a 1 to 3 log decrease in B. thailandensis Bt6  evidence of inhibition, with a complete or partial zone of inhibition around the B. pseudomallei colony and mounding of B. thailandensis growth surrounding this zone  defined by method (i), and as before each B. pseudomallei isolate gave a consistent inhibition result to each of the five B. thailandensis isolates . We tested the cell-free supernatants of sixty-seven lates 55.% definedB. pseudomallei isolates. When cell-free supernatant of B. pseudomallei was spotted onto swarm agar, left to dry for 15 min and then overlaid by B. thailandensis cells (method iii), we observed two outcomes; B. thailandensis failed to swarm when overlaid on supernatant from the same thirty-seven B. pseudomallei, while in the remainder B. thailandensis swarmed without evidence of inhibition . This result suggests that the secreted factor (denoted here as “inhibitory factor”) does not function as growth inhibitor but only inhibited B. thailandensis swarming.Since the supernatant of B. pseudomallei multilocus sequence type (ST) was known for sixty-six out of sixty-seven isolates. The frequency of inhibition was defined for each ST, which demonstrated an association between the two groups. Inhibition was observed in 14 STs represented by 36 isolates, but not in 10 STs represented by 30 isolates. However, 5 STs contained both inhibitory and non-inhibitory isolates .The B. thailandensis motility on swarm agar, we did not find any significant difference between clinical and environmental isolates. Inhibition was found in 20 of 39 clinical (51.3%) and 17 of 28 environmental isolates (60.7%) (p=0.44). The geographical origin of B. pseudomallei demonstrating inhibition included Thailand  and Australia  (p=0.32).By comparing the effect on B. pseudomallei in a single soil sample, with a predominant genotype co-existing with two or three other genotypes [B. thailandensis motility by cell-free B. pseudomallei supernatant for each of the STs identified from 3 independent soil samples [We previously demonstrated genetic diversity of enotypes . We hypo samples   against itself and 66 B. pseudomallei other isolates  were tested against each of five B. thailandensis strains  using the inhibition assay method (ii). B. pseudomallei strains 153, 164 and K96243 were clinical isolates and B3 and B4 were soil isolates. All three colony morphotypes of strains 153, 164 and B4 showed inhibition, whilst all three types of K96243 and B3 did not. This result indicated that the secretion of inhibitory factor by B. pseudomallei is strain-dependent but not related to the morphotypes tested.We previously reported that nditions ,13. We nB. pseudomallei inhibitory factor was growth-phase dependent, we cultured 1 × 105 cfu/ml B. pseudomallei 1710a in 10 ml LB broth at 37°C with shaking at 200 rpm for 24 h. The supernatant was collected every 2 h and filter-sterilized, and these samples examined for the presence of inhibition of B. thailandensis E264 swarming using inhibition assay method (ii). Inhibitory activity was detected in all aliquots collected after 12 h incubation when the B. pseudomallei count reached 5 × 109 cfu/ml and was in stationary phase  were tested. Following filtration, 15 times concentrated volume of original supernatants were obtained and 100 μl filtrate and retentate fractions were tested for the inhibition of B. thailandensis swarming using assay method (ii). The inhibition of B. thailandensis E264 swarming was detected in retentate samples from all membranes. The inhibition was not detected in the filtrate of both B. pseudomallei supernatants when 10 kDa and 30 kDa membranes were used, but was present in filtrate fractions from 50 kDa and 100 kDa membranes  was used to estimate the molecular weight of the inhibitory factor from B. pseudomallei 1710a on B. thailandensis E264 swarming, comparing the activity before and after protein digestion with each of two protease enzymes. The results shown in Figure Next, we determined whether the inhibitory activity was mediated by a protein or non-protein factor. We tested the 15x-concentrated cell-free supernatant of B. pseudomallei 1710a against swarming of B. thailandensis E264 was retained in samples exposed to pH 4, 5, 6 and 7 but disappeared at pH 3 and 8, suggesting that the factor was inactivated at extreme pH . Incubation of supernatant at–80°C, 4°C, 25°C, 37°C and 50°C had no effect while the activity was lost when the sample was incubated at 80°C. The data indicates that the inhibition activity is heat-labile  had reduced motility compared to that after exposure to supernatant from B. pseudomallei K96243 (non-inhibitory strain), B. thailandensis E264, or the unexposed control , compared with 77%, 81% and 95% of B. thailandensis E264 cells exposed to supernatant of B. pseudomallei K96243, B. thailandensis E264 and non-exposed control, respectively  to determine the proportion of flagellated bacteria. Following exposure to cell-free supernatant of fliC gene in B. thailandensis E264 exposed to cell-free supernatant of B. pseudomallei 1710a, B. pseudomallei K96243, B. thailandensis E264 and non-exposed control. No difference in gene expression was observed  coding for inhibitory factor and overexpression of the factor will be necessary for further characterization.The observation that DI types  suppressB. pseudomallei [B. pseudomallei during the rainy season may involve motility [B. pseudomallei mutants defective in the structural flagellin protein (FliC), do not swarm on agar [B. thailandensis was inhibited by half of our B. pseudomallei isolates. Our microscopy results and RT-PCR data suggested that the inhibitory secreted product did not down-regulate flagellar expression, but led to structural changes. The mechanism is unknown but could involve several potential processes such as assembly, secretion, proteolytic degradation and depolymerization.Studies in Lao PDR and Australia have revealed that soil collected in different seasons contain different numbers of domallei ,15. Envimotility ,16. Flag on agar . Here, wB. pseudomallei genotype. In contrast, we did not find a difference in inhibitory effect on B. thailandensis swarming motility for three isogenic morphotypes from each of five B. pseudomallei isolates, even though a previous study demonstrated that colony morphology variation represents several phenotypic differences [Analysis of genotype showed that the flagellar inhibitory factor was present in all of the isolates tested from 14 STs, and was absent in all isolates from 10 other STs, although 5 STs were present in both groups. Our test isolates included the two most abundant STs in Thailand (ST70 and ST54), which reside in different clonal complexes , and exhferences .B. pseudomallei population structuring within a single soil sample is that the predominant clone may have a greater ability to inhibit B. thailandensis and minor populations of B. pseudomallei. We demonstrated that all B. pseudomallei isolates with the highest genotype frequency for each of three soil samples consistently inhibited motility of B. thailandensis. However, some minor populations also had this ability. The finding that the culture supernatant of all B. pseudomallei did not inhibit motility of any other B. pseudomallei suggests that the role of this secreted factor may be to inhibit the motility of other species. Flagellar filament of bacteria is composed of a flagellin subunit, which is diverse among bacterial species [B. pseudomallei and B. thailandensis. However, sequence alignment between several isolates of the two species has revealed a consistent 5 amino acid deletion at positions 247–251 (SPSFQ) in the flagellin of B. thailandensis [B. pseudomallei and 300 or 342 Da for B. thailandensis) [B. pseudomallei.One explanation for  species . Bioinfoandensis . In addindensis) . It is pB. pseudomallei to survive in diverse environments is likely to be associated with a genetic repertoire that facilitates competition and adaptation [B. pseudomallei strains [B. pseudomallei culture supernatants had no effect on growth of B. thailandensis. Previous work demonstrated that B. pseudomallei CdiA-CT toxins are functional [B. pseudomallei exhibits tRNase activity, which blocks the growth of B. thailandensis E264 target cells [B. pseudomallei [The ability of aptation . This in strains . Our resnctional . For exaet cells . It is adomallei .B. pseudomallei and B. thailandensis. Inhibition of B. thailandensis by B. pseudomallei 1710a was only observed at a ratio greater than 1:1, suggesting that the B. pseudomallei inhibitor may mediate growth inhibition in a cell-density dependent or in a co-operative manner. Such cooperation has not been reported for B. pseudomallei, but it has been reported that the CDI genes in B. thailandensis were expressed in a subpopulation during culture in broth and also played a role in biofilm formation [Our analysis suggests that this growth inhibition phenomenon was not the result of different growth rates of ormation ,23.B. pseudomallei inhibits both the growth and motility of its close relative B. thailandensis. The latter phenomenon appears to occur via a previously unreported mechanism involving an effect on flagellar structure.Our results indicate that B. pseudomallei and six B. thailandensis isolates were examined in this study. The B. pseudomallei isolates originated from Thailand (thirty-six from human infection and twenty-seven from the environment) or Australia  [B. thailandensis  were isolated from soil in central Thailand and the sixth isolate was B. thailandensis Bt6, a kanamycin resistant mutant derived previously from B. thailandensis E264 (E264 attTN7::miniTn7T-Kan) [B. pseudomallei strains were tested, which were derived from five isolates  [B. thailandensis Bt6. The study was granted exemption from requiring ethics approval by Ethics Committee of Faculty of Tropical Medicine, Mahidol University.Sixty-seven l water) ,24 (Addin7T-Kan) . A furthhailand)  (Additiohailand) ,13. Bact600 of 0.15 to obtain 1 × 108 cfu/ml. One hundred microlitres of bacterial suspension was added to 10 ml of LB broth and incubated at 37°C in air with shaking at 200 rpm for 24 h. At 2 h intervals, 100 μl of bacterial culture was collected, serially diluted 10-fold in PBS, and the number of viable cells counted by plating on LB agar or LB agar containing 500 μg/ml Kan (for B. thailandensis Bt6) in triplicate. Plates were incubated at 37°C in air for 2 days and doubling time was calculated.A single bacterial colony was suspended in sterile phosphate buffered saline (PBS) and adjusted to an ODB. pseudomallei on growth of B. thailandensis. B. pseudomallei supernatant from overnight culture in LB broth at 37°C was concentrated using a filter membrane with 10,000 cut-off  to 5×, 2.5× and 1.2×, and an equal volume of each concentrate added to 50 μl of an overnight culture of B. thailandensis to obtain a final concentration 5 × 105 cfu/ml in LB. The control was performed by adding an equal volume of PBS to 50 μl of an overnight culture of B. thailandensis. The experiment was performed in a 96-well plate in triplicate in a given experiment. Cultures were incubated at 37°C in air for 18 h. Bacterial growth was defined as visual turbidity followed by colony count.A broth microdilution assay was used to determine the effect of cell-free supernatant from B. pseudomallei and B. thailandensis affected swarming motility. (i) A colony of B. pseudomallei and a colony of B. thailandensis obtained from an overnight culture on LB agar were spotted using a pipette tip at opposite poles of a swarm agar plate, and the growth observed daily over a 3 day incubation time course at 37°C. (ii) Supernatant from an 18 hour B. pseudomallei culture in LB broth was harvesting using centrifugation and filtrated through 0.2 μm membrane . A colony of B. thailandensis from overnight culture on LB agar was spotted onto the center of the plate and incubated at 37°C for 16 h, after which 100 μl of cell-free B. pseudomallei supernatant was dropped onto the plate at 2, 6 and 10 o’clock and 1–1.5 cm from the edge of the plate. The plate was further incubated for 16 h, and then observed for inhibition of B. thailandensis swarming, which was defined as the presence of any clear zone around the supernatant drop. We spotted the supernatant of B. pseudomallei after B. thailandensis was allowed to swarm for a period of time because the inhibition activity was lost if applied at the beginning of the assay. (iii) 100 μl of cell-free B. pseudomallei supernatant was dropped in the center of a swarm plate and left to diffuse and dry in air for 15 min. The same spot was then inoculated with a B. thailandensis colony using a pipette tip, and the plate then incubated for 18 h before the swarm B. thailandensis colony was examined.Three methods were devised to test whether interactions between B. pseudomallei mixed with B. thailandensis in a ratio of 1000:1, 100:1, 10:1 or 1:1, and 100 μl of the mixture dropped onto a 2.5 × 2.5 cm2 sterile nitrocellulose membrane which was placed on top of LB agar. Plates were incubated at 37°C in air for 24 h after which the nitrocellulose membrane was removed, placed into 10 ml PBS and vortexed vigorously for 30 sec to harvest bacteria. Duplicate plating onto LB and LB plus Kan was performed, and plate counts determined for B. thailandensis Bt6 (LB/Kan plate) and B. pseudomallei . To examine the effect of incubation time, a co-culture of B. pseudomallei 1710a and B. thailandensis Bt6 at a ratio of 1000:1 was incubated at 37°C for 3 h, 6 h and 24 h. At the indicated time point, cells were harvested from the plates and viable bacteria counted as before. The interaction was also tested between B. thailandensis Bt6 and six B. pseudomallei isolates , in which B. thailandensis E264 wild type was used for comparison using a ratio of B. thailandensis E264 to B. thailandensis Bt6 at 1000:1 for 24 h. Three B. pseudomallei isolates possessed different CDI types  [A growth inhibition assay was carried out as described previously . In brieB. pseudomallei was inoculated into LB and incubated overnight at 37°C in air with shaking at 200 rpm. The bacterial culture was centrifuged at 4,000 × g for 15 min at 4°C and the supernatant filtrated through a 0.2 μm membrane. To estimate the molecular size of the inhibitory substance, 10 ml of the cell-free supernatant of B. pseudomallei 1710a was filtrated through filter membranes with a molecular weight cut-off (MWCO) of 10,000, 30,000, 50,000 or 100,000 daltons  to obtain 15–100 times concentrated culture supernatant. The presence of inhibitory substance in the retentate or filtrate after membrane separation was assessed using the inhibition motility assay (method (ii)).B. pseudomallei 1710a was concentrated 15 times using 30,000 membrane filtration, quantified using the bicinchoninic acid assay , and analyzed by SDS-PAGE [2 at 37°C for 72 h).The protein nature of the inhibitory factor was characterized by digestion with two different proteases. Cell-free supernatant of 18 h culture at 37°C in LB broth of SDS-PAGE . The samB. pseudomallei 1710a was tested after adjustment to a pH of 3, 4, 5, 6, 7 or 8, and after being maintaining at pH 7.0 at –80°C, 4°C, 25°C, 37°C, 50°C or 80°C for 24 h. Inhibition activity was also tested at pH 7.0 in the presence of NaCl at a final concentration of 0.2 M, 0.4 M, 0.6 M, 0.8 M or 1 M. Aliquots of 100 μl were tested in the B. thailandensis E264 inhibition motility assay at 37°C for 18 h (method ii). Untreated overnight cell-free supernatant of B. pseudomallei 1710a in LB broth was used as a positive control and LB broth was used as a negative control. Results for inhibition of B. thailandensis motility were read based on the presence (positive) or absence (negative) of a clear zone on swarm agar.Inhibition activity of cell-free supernatant of B. thailandensis E264 after exposure to cell-free supernatant from B. pseudomallei 1710a, B. pseudomallei K96243 and B. thailandensis E264 (control). The method was modified from a previous study [B. thailandensis cells were exposed to B. pseudomallei cell-free supernatant using the swarming inhibition motility method (iii). B. thailandensis E264 was picked from the colony at the end of this assay and suspended in 50 μl PBS. Two microlitres of bacterial suspension was overlaid on a film of 1.5% low-melting agarose pad  on a microscope glass slide. A cover slip was placed and motility was observed by light microscope  with 100× magnification. Video was recorded and 20 individual cells were tracked and analyzed for 20 sec motility using ImageJ program (http://rsb.info.nih.gov/ij/) with the manual tracking plug-in.Live cell imaging was used to examine the motility of B. thailandensis E264 cell preparation was examined using TEM for the presence of flagella. Fifty microliters of the B. thailandensis suspension was dropped onto parafilm and Fomvar coated carbon grids were placed on top for 10 min to transfer bacterial cells. The liquid was carefully removed with filter paper and the samples were stained with 1% uranyl acetate for 10 min after which the liquid was again carefully removed. The grid was dried at room temperature for overnight. Bacteria were observed with a Hitachi Electron Microscope H-7000 (Japan). The presence of bacterial flagella was recorded for 100 bacteria per isolate.The same B. thailandensis E264 cells were picked from a colony at the completion of method (iii) as above. RNA was extracted using Trizol reagent, as described previously [fliC of B. thailandensis E264 (accession number AF081500.1) using Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast). One-step reverse transcriptase (RT)-PCR was performed using 1 μg RNA and a Superscript III One-step RT-PCR system  with forward primer 5′ GGTCGCTCAACAGAACCTCA 3′ and reverse primer 5′ CTGGTTCAGGCCGTTGATCT3′. The RT-PCR conditions were as follows: cDNA synthesis at 45°C for 30 min; initial denaturation at 95°C for 5 min; 30 cycles of denaturation at 94°C for 30 sec, annealing at 50°C for 15 sec, and extension at 72°C for 30 sec; and a final elongation step at 72°C for 7 min. The positive control was RT-PCR for 23S rRNA amplification using primers 23S_F and 23S_R with forward primer 5′ GTAGACCCGAAACCAGGTGA3′ and reverse primer 5′ CACCCCTATCCACAGCTCAT 3′. The negative control was a reaction without RT enzyme. The amplified product was run on a 1.5% agarose gel, stained with ethidium bromide and visualized under UV light.eviously . PrimersStatistical analyses were performed using Stata, version 12 . Fisher’s exact test or Chi square tests were used to test proportions. One-way ANOVA was used to test the difference between groups. Quantitative data are presented as mean ± standard deviation. Differences were considered statistically significant at a p-value ≤ 0.05.


Papaverine is a nonspecific inhibitor of phosphodiesterases, leading to increases in both intracellular cGMP and cAMP. We hypothesized that papaverine reduces force by decreasing intracellular calcium concentrations ([Ca2+]i levels were concurrently measured. Filamentous actin (F-actin) level was measured by an in vitro actin assay. Tissue was snap frozen to measure myosin light chain and actin regulatory protein phosphorylation. Pre-treatment with papaverine completely inhibited norepinephrine-induced force generation, blocked increases in [Ca2+]i and led to a decrease in the phosphorylation of myosin light chain. Papaverine pre-treatment also led to increased phosphorylation of the heat shock-related protein 20 (HSPB6) and the vasodilator stimulated phosphoprotein (VASP), as well as decreased filamentous actin (F-actin) levels suggesting depolymerization of actin.HSV was equilibrated in a muscle bath, pre-treated with 1 mM papaverine followed by 5 μM norepinephrine, and force along with [Ca2+]i-mediated inhibition of myosin light chain phosphorylation and actin regulatory protein phosphorylation-mediated actin depolymerization. Thus, papaverine induces sustained inhibition of contraction of HSV by the modulation of both myosin cross-bridge formation and actin cytoskeletal dynamics and is a pharmacological alternative to high pressure distention to prevent vasospasm.These results suggest that papaverine-induced force inhibition of HSV involves [Ca The human saphenous vein (HSV) is the most commonly used conduit for coronary artery bypass grafting (CABG). However, HSV is regarded as inferior to the internal mammary artery (IMA) because of 50% failure rate within 10 years compared with 5% failure for the IMA. Saphenoex vivo prior to implantation of the HSV has been the most commonly used approach for preventing vasospasmi concentration through calcium regulatory proteins [2+]i and activation of myosin light chain phosphatase 1, which reduces the sensitivity of the contractile apparatus to intracellular calcium[2+]i have been proposed to involve several mechanisms including activation of Ca2+ -ATPase in the plasma membrane and sarcoplasmic reticulum[2+-activated K+ channels[2+-ATPase[2+ release by the sarcoplasmic reticulum[2+ -activated K+ channel [2+]i. A decrease in intracellular [Ca2+]i causes inactivation of myosin light chain kinase, dephosphorylation of myosin light chain by the myosin phosphatase, which causes a reduction in the sensitivity of the contractile apparatus to [Ca2+]i hav hav2+]i o [Ca2+]i,27,28,29o [Ca2+]i and by ao [Ca2+]i.2+]i and calcium sensitivity of the contractile apparatus, regulation of actin cytoskeletal dynamics have been implicated during relaxation or inhibition of contraction mediated by vasodilators -1-propanesulfonate (CHAPS) were obtained from Research Organics Inc. . F/G Actin assay kit was obtained from Cytoskeleton Inc., . Fura 2-AM and Pluronic F-127 were obtained from Invitrogen . All other chemicals were obtained from Sigma Chemical Co.  unless specified otherwise.4, 1.0 mM NaH2PO4, 10 mM glucose, 1.5 mM CaCl2, and 25 mM Na2HCO3, pH 7.4), equilibrated with 95% oxygen and 5% carbon dioxide at 37°C. Each ring was progressively stretched to its optimal resting tension (approximately 1 g) that would produce a maximal response to contractile agonists as determined previously, then maintained at the resting tension and equilibrated for a minimum of 2 hours  = force (g) x 0.0987 / area, where area is equal to the wet weight [(mg) / length ] divided by 1.055),. Br. Br2+ meProteins from frozen muscle rings were extracted in UDC buffer , 4% CHAPS containing protease inhibitors, and Phosphatase inhibitor I and II . The mixtures were vortexed at room temperature overnight, and then centrifuged at 14,000 rpm for 15 min at 4°C. Soluble protein concentrations were determined using the Coomassie Plus protein assay . Equal amounts (20–50 μg) of proteins were placed in a Laemmli sample buffer , heated for 5 min at 100°C and separated on SDS polyacrylamide gels. Proteins from the gels were transferred onto nitrocellulose membranes  and blocked prior to incubation overnight at 4°C with the following primary antibodies: anti-HSPB6 ; anti-VASP ; anti-MYPT1 ; anti-pMYPT1 (thr 696) ; anti-MLC20 . Membranes were washed three times with TBS containing Tween 20 (0.1%) (TBS-T), and incubated with appropriate infrared-labeled secondary antibodies  for 1 h. at room temperature. The membranes were again washed with TBS-T, and protein-antibody complexes were visualized and quantified using the Odyssey direct infrared fluorescence imaging system (Li-COR). Phosphorylation was calculated as a ratio of the phosphorylated protein to total protein and was then normalized to the unstimulated control with the control value set as 1.0.-6M) for 5 min or pre-treated with papaverine (10−3 M) for 10 min, followed by norepinephrine for 5 min, and snap frozen as described above. Myosin light chain phosphorylation was determined using a modification of an established method described earlier i, and since myosin light chain (MLC20) phosphorylation correlates with changes in [Ca2+]I, papaverine pretreatment should prevent increases in MLC20 phosphorylation. Norepinephrine treatment significantly increased the phosphorylation of MLC20 (0.382 ± 0.06 Mol Pi/Mol MLC20) when compared to untreated basal (0.11 ± 0.03 Mol Pi/MolMLC20) . Pretreatment with papaverine followed by norepinephrine treatment significantly inhibited the phosphorylation of MLC20 (0.14 ± 0.04 Mol Pi/Mol MLC20) when compared to norepinephrine alone , pretreated with papaverine  followed by norepinephrine , or papaverine alone , and changes in actin polymerization was analyzed by measuring F and G-actin level using a F/G actin assay. Treatment with norepinephrine led to increases in F-actin by 13% , while treatment with papaverine reduced F-actin by 12% . Pretreatment with papaverine for 10 min before norepinephrine stimulation reduced the F- actin by 29% . We detp < 0.05, n = 4)] in HSV. HSPB6 remained phosphorylated even after norepinephrine was added to induce contraction ] in HSV. MYPT1 phosphorylation had had a non-significant decrease in tissue pretreated with papaverine and then treated with norepinephrine  during inhibition of contraction or force suppression. The decreases in force and [Ca2+]i were correlated with decreased myosin light chain phosphorylation levels which demonstrates that papaverine inhibits myosin cross bridge phosphorylation (In this study, a physiologic system was developed in which vasospasm of HSV was induced by treatment with norepinephrine and the mechanism of papaverine-induced prevention of vasospasm was examined. Treatment of HSV with papaverine resulted in complete suppression of force generation in the presence of a contractile agonist, norepinephrine . This phrylation .et al have reported that HSPB6 phosphorylation causes relaxation through depolymerization of F-actin as well as inhibition of myosin binding to actin I and myosin light chain phosphorylation, suggesting thick filament mechanism of force inhibition. In addition inhibition of force production was associated with increases in the phosphorylation of the actin regulatory proteins HSPB6 and VASP and decreases in filamentous actin suggesting actin cytoskeletal mechanism of force inhibition. Thus, papaverine likely inhibits vasospasm through multiple regulatory processes. As data emerges describing the cellular harm caused by manual distention[Treatment of HSV with papaverine inhibits force production by norepinephrine. This inhibition of force production was associated with inhibition of [Caistention, papaver


Cerebral ischemia and subsequent reperfusion induced dephosphorylation and phosphorylation of 4E-BP2 at Thr37/Thr46, respectively. The induced phosphorylation was in parallel with the release of 4E-BP2 from eIF4E, although two of the phosphorylated 4E-BP2 forms were bound to eIF4E. Upon long-term reperfusion, there was a decrease in the binding of 4E-BP2 to eIF4E in cerebral cortex, demonstrated by cap binding assays and 4E-BP2-immunoprecipitation experiments. The release of 4E-BP2 from eIF4E was without changes in 4E-BP2 phosphorylation or other post-translational modification recognized by 2-DGE. These findings demonstrated specific changes in 4E-BP2/eIF4E association dependent and independent of 4E-BP2 phosphorylation. The last result supports the notion that phosphorylation may not be the uniquely regulation for the binding of 4E-BP2 to eIF4E under ischemic stress.Eukaryotic initiation factor (eIF) 4E-binding proteins (4E-BPs) are translational repressors that bind specifically to eIF4E and are critical in the control of protein translation. 4E-BP2 is the predominant 4E-BP expressed in the brain, but their role is not well known. Here, we characterized four forms of 4E-BP2 detected by two-dimensional gel electrophoresis (2-DGE) in brain. The form with highest electrophoretic mobility was the main form susceptible to phosphorylation at Thr Ischemia induces a period of hypoxia and energy depletion inducing an inhibition of translational rates . Normal 7GpppN ; and eIF4G, a scaffolding protein that provides docking sites for the aforementioned initiation factors [37/Thr46 on 4E-BP1 appears to be the critical step because it is the priming event for subsequent (hyper)phosphorylation and prevention of the binding to eIF4E in cell lines [The translation process has an important control point in the recruitment of the 40S ribosomal subunit to the 5' end of mRNA. In this process, a key step is the assembly of eukaryotic initiation factor (eIF) 4F complex, which contains eIF4A, an ATP-dependent RNA helicase; eIF4E, which binds to the mRNA 5'-cap structure m factors . The bes factors , 7. The ll lines .cornu ammonis 1 (CA1) region. Cerebral cortex and CA1 were used as resistant and vulnerable regions, respectively, against global cerebral ischemia [Previously, we have reported dephosphorylation of 4E-BP1 after ischemia and induction of 4E-BP1 phosphorylation during ischemic reperfusion . These cischemia , 11. In 37 and/or Thr46 of the human sequence) and anti-phospho-ribosomal protein S6 (rpS6) (#4858) (Ser235/Ser236) antibodies were from Cell Signaling. Mouse monoclonal anti-eIF4E antibody (610269) was from BD Transduction Labs and anti-rpS6 antibody (#2317) was from Cell Signaling. Goat polyclonal anti-eIF4G antibody  was from Santa Cruz Biotechnology. Rabbit polyclonal anti-β-tubulin antibody (PRB-435P) was from Covance. The chemicals used in isoelectric focusing and SDS-PAGE were purchased from Bio-Rad and GE Healthcare. All general chemicals were purchased from Sigma unless stated otherwise.Rabbit polyclonal anti-4E-BP2 (#2845), anti-phospho-4E-BP1/2 (#9459)  by the standard four-vessel occlusion model described previously . BrieflyThe cerebral cortex and hippocampal CA1 region from control, ischemic and IR animals were rapidly dissected out under a magnifying glass. The samples were homogenized 1:5 (w/v) with buffer A , as described previously , 14. The7GTP)-Sepharose—was used [7GTP-Sepharose 4B  and incubated for 30 min at 4°C in buffer A with 100 mM potassium chloride and 100 μM GTP. The beads were centrifuged at 2500 × g for 5 min and washed in the same buffer three times. The proteins were removed from m7GTP-Sepharose with SDS loading buffer and subjected to SDS-PAGE or two-dimensional gel electrophoresis and western blotting. The immunoblots were developed separately with the antibodies described above against eIF4G, eIF4E and 4E-BP2 and quantified as described below.In order to study eIF4F complex formation and the binding of 4E-BP2 and eIF4G to eIF4E, a cap-containing matrix—7-methyl-GTP  of each experimental condition were incubated with 2 μg of rabbit polyclonal anti-4E-BP2 antibody (Sigma) overnight and then further incubated with Protein G-Agarose 4 (ABT) for 1 h at 4°C on rotating shaker. Immunoprecipitated proteins were then washed 3 times in lysis buffer and subsequently subjected to SDS-PAGE or two-dimensional gel electrophoresis and western blotting.7GTP-Sepharose or 4E-BP2 immunoprecipitates of each different experimental condition were analyzed by SDS-PAGE   or two-dimensional gel electrophoresis (see below) and transferred onto PVDF membranes . The membranes were incubated for 1 h at room temperature or overnight at 4°C with the antibody against the specific protein to be detected, washed, then incubated for 1 h with peroxidase-conjugated anti-mouse,-rabbit  or-goat (Santa Cruz Biotechnology) IgG, and developed with ECL reagent . For phospho-protein detection, the blots were probed with the phospho-specific antibody, stripped, and reprobed with the corresponding anti-total protein antibody. Phospho-4E-BP2 and 4E-BP2 were also analyzed in twin separate experiments to avoid the potential cross-reaction between the different rabbit polyclonal antibodies. The western blots were quantified using ImageQuant TL software . Internal standards (tubulin) were included to normalize the different immunoblots. Data of the phospho-forms or phospho-proteins were expressed in arbitrary units with respect to the levels of total protein (ratios). Protein markers (range: 12–225 kDa)  were used to calculate the apparent molecular weight (MW).Samples of PMS (35 μg), m7GTP-Sepharose of each experimental condition were added to 8.5 M urea/5% β-mercaptoethanol (Bio-Rad) and loaded into horizontal IEF slab gels as the first dimension. IEF was performed with immobilized pH 3‒10 nonlinear gradient  (Bio-Rad) strips (10 cm) in a flatbed Multiphor II Electrophoresis System , according to the manufacturer’s instructions. The first dimension was combined with standard vertical slab SDS-PAGE as the second dimension   performed in 1.0 mm thick gels with the IEF strip used as stacking gel, as described previously [Samples of PMS (75 μg) or meviously , 18; andIn other experiments, immunoprecipitated samples were labeled on their lysine residues with Cy3 and Cy5 minimal dyes . Pairs of experimental and control samples were mixed and analyzed by 2-DGE as described above. The fluorescent gels were scanned using a Typhoon 9200 imager .t test to compare the data respect to the SHC or SHC3d control groups. Comparisons between the cerebral cortex and hippocampal CA1 regions were performed using Newman-Keuls post-test after ANOVA, when was significant, or by paired t test. Statistical significance was set at p < 0.05 using Prism statistical software (GraphPad Software).The different animals from each experimental condition were independently analyzed in duplicate and their averaged values were used for statistical analysis. Data were represented in arbitrary units and expressed as mean ± SEM of the 4–8 different animals. Statistical analysis was performed either using ANOVA following Dunnett’s post-test, when was significant, or by Student’s a and b forms in order of decreasing electrophoretic mobility , a downstream target sensitive to mTOR activity . The phon in R30 .; wherean in R30 ..a’, that corresponded to the a form of 4E-BP2; and three spots, b’, b” and b”’  that corresponded to the b form  of 4E-BP2 (computed in http://www.bioinformatics.org/sms2/). R30 induced two additional spots detected in the a form, a” and a”’ (pI = 5.67 and 5.15), and one additional spot between b” and b”’, b”” (pI = 5.3)  and rabbit polyclonal anti-phospho-4E-BP1 (Thr70) antibodies  were probed and no signal was detected on 4E-BP2 .To characterize further the physiological 4E-BP2 (phospho)forms induced by ischemia and upon reperfusion, we analyzed 4E-BP2 by two-dimensional gel electrophoresis and western blotting. The study was performed in cortical samples as no significant differences were found between the cerebral cortex and the CA1 region (see above). In the first dimension, pH 3–10 strips with a nonlinear gradient were used which improved the resolution in the pH 5–6.5 region that corresponded to the theoretical pI range of 4E-BP2. Four different spots were detected in all samples: a most basic spot (pI = 6.4), e b form . The pI I = 5.3) , R30. NoI = 5.3) . Phospho”’ spots . These rup Figs.  and 2. S7GTP-Sepharose, a cap-containing matrix [a and b were bound to eIF4E . Therefore, in R30, the binding of 4E-BP2 to eIF4E decreased in parallel with the phosphorylation of 4E-BP2, although a fraction of the phosphorylated 4E-BP2 was found bound to eIF4E.The eIF4E is identified by its ability to bind to the 5'-cap structure and was consequently isolated by affinity chromatography in mg matrix . Using tto eIF4E , 4E-BP2.to eIF4E . 4E-BP2 a”’ spot , R30. Nom in R30 , a condim in R30 . The pho7GTP-Sepharose. The results showed that identical a’, b’, b” and b”’ forms of 4E-BP2 were bound to eIF4E in the SHC3d control or R3d in both cerebral cortex and CA1 region, with no differences between them  induced a significant decrease in the binding of 4E-BP2 to eIF4E in the cerebral cortex compared with SHC3d control . Decreaseen them . These reen them . 4E-BP2 een them . All thed 4E-BP2  and abled 4E-BP2 . In summ37/Thr46 in R30, and not in R3d phosphorylated 4E-BP2 and the a form would be un- or hypo-phosphorylated 4E-BP2. To confirm this, we studied the phosphorylation at Thr37/Thr46 sites. Unlike 4E-BP1, 4E-BP2 phosphorylation on Thr37/Thr46 was poorly detected in the b form, and was mainly detected at the higher mobility form in two spots, a” and a”’, without changes in the apparent molecular weight of 4E-BP2. So far, the results showed two forms of 4E-BP2, one with a lower electrophoretic mobility and another with higher electrophoretic mobility that was susceptible to phosphorylation at Thr37/Thr46 sites. Phosphorylation that was resolved into more acidic spots by 2-DGE. Ischemia and subsequent reperfusion induced dephosphorylation and phosphorylation of 4E-BP2, respectively. This phosphorylation was restored to control levels upon long-term reperfusion.Because 4E-BP1 in brain is resolved in three bands with decreasing electrophoretic mobility that correspond with increasing phosphorylation status , 16, the37/Thr46, in parallel with a decrease in the 4E-BP2 bound to eIF4E and a significant increase in the binding of eIF4G to eIF4E. Further, the more acidic form of phosphorylated 4E-BP2 did not bind to eIF4E. The induced 4E-BP2 phosphorylation was correlated with the increased phosphorylation of rpS6 at Ser235/236, a sign of mTORC1 activity. These results agree with the mTORC1-dependent 4E-BP1 phosphorylation at Thr37/Thr46 [IR stress during short-term reperfusion (R30) induced phosphorylation of 4E-BP2 at Thr37/Thr46 , and wit37/Thr46 .37/Thr46 sites; (iii) this phosphorylation did not induce any shifts in their apparent molecular weight; (iv) all detected unphosphorylated forms of 4E-BP2 bound to eIF4E; and (v) 4E-BP2 phosphorylation at Thr37/Thr46 allowed the binding to eIF4E. The last result was confirmed in primary neuronal cultures subjected to ischemia in vitro, where phosphorylation at Thr37/Thr46 was detected in the 4E-BP2 bound to eIF4E  4E-BP2 was resolved in SDS-PAGE into forms that are apparently independent of phosphorylation; (ii) the highest migrating form was the principal 4E-BP2 form susceptible to phosphorylation at Thrto eIF4E .. However 4E-BP1 .65 and Thr70—were not detected on 4E-BP2  during long-term reperfusion in the cerebral cortex compared with the control condition or CA1.Interestingly, long-term reperfusion (R3d) induced a decrease in the 4E-BP2 bound to eIF4E in the cerebral cortex without increase in their phosphorylation levels or changes in the 4E-BP2 forms, as demonstrated the 2-DGE experiments. The dissociation of 4E-BP2 and eIF4E during long-term reperfusion was also demonstrated by affinity to the cap-containing matrix (this paper), in 4E-BP2 immunoprecipitates (this paper), and was in agreement with immunohistochemical studies in brain sections and in parallel to the increase of translational rates . Besides209 site [209) antibody . No significant changes in eIF4E phosphorylation were found in R3d between the cerebral cortex and CA1 at R3d or when compared with the control (not shown). We also consider eIF4GI and II as potential interaction factors. We studied eIF4GI and II levels, and we did not find differences between the cerebral cortex and CA1 or controls (not shown). In addition, we studied the phosphorylation of eIF4G, but only anti-phospho-Ser1108, anti-phospho-Ser1186, and anti-phospho-Ser1232 antibodies are available against rat, whereas several and multiple phosphorylation sites are described in human eIF4GI and II [We study if eIF4E –the other partner in 4E-BP2/eIF4E association—was implicated in ischemia and IR stress, or correlated with 4E-BP2/eIF4E association. We analyzed the phosphorylation levels at the regulated Ser209 site  with a rI and II . To dateThe results present here show the (hyper)phosphorylation of 4E-BP2 and the inhibition of the binding to eIF4E upon reoxygenation after ischemia (R30), when mTOR signaling is activated . InteresS1 Fig37 and/or Thr46 antibody  in the absence (left panel) or presence (right panel) of phospho-4E-BP1 (Thr37/Thr46) blocking peptide , and re-probed with anti-4E-BP2 (lower panel) antibody. (B) The membrane was probed with anti-phospho-4E-BP1/2 Thr37 and/or Thr46 , and re-probed with anti-4E-BP1 (middle panel) and anti-4E-BP2 (lower panel) antibodies to exact identification. The results show that β and γ forms of 4E-BP1 and “a” and “b” forms of 4E-BP2 are susceptible to detection by the anti-phospho-Thr37/Thr46 antibody in rat brain. Under the electrophoretic conditions of this work , “beta” and “gamma” forms of 4E-BP1 are resolved at 20 and 21 kDa, respectively, whereas “a” and “b” forms of 4E-BP2 are resolved at 17 and 18.5 kDa, respectively. These differences are sufficient to distinguish one from another. The right numbers indicate the apparent molecular weight (MW) in kDa from protein markers.Control and ischemic-reperfusion brain samples were subjected to SDS-PAGE and western blot. (A) The membrane was probed with anti-phospho-4E-BP1/2 Thr(TIF)Click here for additional data file.S2 Fig235/236 (p-rpS6) and anti-rpS6 (rpS6) antibodies. Arrows indicate the detected phospho-rpS6 and rpS6. The right numbers indicate the apparent MW in kDa from protein markers. No significant differences in the rpS6 levels were found . Data (bar graph) are the quantification of phospho-rpS6 with respect to total rpS6 levels (ratios) from four to six different animals run in duplicate and represented in arbitrary units; error bars indicate SEM. *p < 0.05, compared with the controls.Samples of the cerebral cortex, C, or hippocampal CA1 region, CA1, from control (SHC and SHC3d) and ischemic animals, without (I15) or with reperfusion (R30 and R3d), were subjected to western blotting with anti-phospho-rpS6 Ser(TIF)Click here for additional data file.S3 Fig37/Thr46 (p-Thr37/46) antibodies. (B) Alternatively, cell lysates were bound to m7GTP-Sepharose and analyzed by western blotting as above described. Arrows show the β and γ positions for 4E-BP1, and the a and b forms of 4E-BP2. Note that phosphorylation at Thr37/Thr46 was detected in the 4E-BP2 bound to eIF4E in the cap-containing matrix (m7GTP-Sepharose), but this phosphorylation was not present for 4E-BP1, as it was described previously [Primary neuronal cells in culture (control)  were subeviously . The rig(TIF)Click here for additional data file.S4 Fighttp://www.uniprot.org/).Identical amino acids between 4E-BP1 and 4E-BP2 are marked in blue and yellow in the human and rat sequence, respectively. Homology between human and rat is marked in bold type. The phosphorylation regulation sites are marked in red; the amino acids susceptible to deamidation in green. The eIF4E binding site  is boxed(TIF)Click here for additional data file.


First proliferating ‘slender' bloodstream forms differentiate to non-dividing ‘stumpy' forms arrested in G1. Second, in response to environmental cues, stumpy bloodstream forms re-enter the cell cycle and start to proliferate as procyclic forms after a lag during which both cell morphology and gene expression are modified. Nearly all arrested cells have lower rates of protein synthesis when compared to the proliferating equivalent. In eukaryotes, one mechanism used to regulate the overall rate of protein synthesis involves phosphorylation of the alpha subunit of initiation factor eIF2 (eIF2α). The effect of eIF2α phosphorylation is to prevent the action of eIF2B, the guanine nucleotide exchange factor that activates eIF2 for the next rounds of initiation. To investigate the role of the phosphorylation of eIF2α in the life cycle of T. brucei, a cell line was made with a single eIF2α gene that contained the phosphorylation site, threonine 169, mutated to alanine. These cells were capable of differentiating from proliferating bloodstream form cells into arrested stumpy forms in mice and into procyclic forms in vitro and in tsetse flies. These results indicate that translation attenuation mediated by the phosphorylation of eIF2α on threonine 169 is not necessary for the cell cycle arrest associated with these differentiation processes.The trypanosome life cycle consists of a series of developmental forms each adapted to an environment in the relevant insect and/or mammalian host. The differentiation process from the mammalian bloodstream form to the insect-midgut procyclic form in  The process includes two differentiation steps in vivo, first proliferating ‘slender' bloodstream forms differentiate to non-dividing ‘stumpy' forms arrested in G1 Kinetoplastids are protozoa and a large number of species have evolved to infect humans and/or animals. Many of the pathogenic species have complex life cycles and have evolved to proliferate in different niches within one or more host through evolution of a series of developmental forms each adapted to an environment in the relevant invertebrate and, sometimes, vertebrate host. The differences between one developmental form and another include alterations in gene expression and cellular morphology. The transition from one developmental form to another has been used to investigate the regulation of these processes Selective transcriptional control by RNAPII is unlikely to play a role in the G1 arrest that occurs on differentiation to stumpy forms. In trypanosomes, genes occur in long tandem arrays, usually encoding functionally unrelated proteins, and are transcribed polycistronically from occasional transcription start sites; co-transcriptional processing results in individual monocistronic mRNAs. This lack of transcriptional regulation of individual genes means that the relative levels of individual mRNAs are determined post-transcriptionally. RNAPII transcription must continue in arrested stumpy cells as some mRNAs have increased levels In eukaryotes, one mechanism used to regulate the overall rate of protein synthesis involves the phosphorylation of serine 51 (S51) in the eIF2α. eIF2 is a trimeric G-protein that forms a ternary complex with the initiator methionyl tRNA that delivers the Met-tRNA to the pre-initiation complex. At the start codon, release of eIF2 occurs after GTP hydrolysis. The effect of eIF2α phosphorylation is to prevent the action of eIF2B, the guanine nucleotide exchange factor that activates eIF2 for the next rounds of initiation. This is turn reduces the amount of available ternary complex and the rate of translation initiation. The kinases that phophorylate eIF2α contain a catalytic domain attached to different regulatory domains. The best conserved are GCN2, which is activated by amino acid deficiency, and PERK which is activated by an increase in unfolded proteins in the ER. In yeast and mammals, eIF2α phosphorylation also initiates changes in gene expression promoted by proteins whose synthesis is paradoxically increased when there is increased eIF2α-P in cells, such as yeast GCN4 and ATF4 in mammals. Both are transcription factors that regulate the expression of hundreds of genes necessary for the cells to recover from the initial stress Trypanosoma sp. and threonine166 in Leishmania sp T. brucei eIF2α T169 is phosphorylated in vitro by eIF2K2, one of the three eIF2α kinases in trypanosome genomes In vivo, this same residue is phosphorylated by eIF2K2 activated by different stress conditions in T. cruzi and Leishmania. In Leishmania, phosphorylation of eIF2α T166 has been shown to be necessary for the normal kinetics of differentiation of promastigotes into amastigotes but not for the process itself T. cruzi, phosphorylation of eIF2α is also required for nutrient-induced differentiation from non-infective epimastigotes into infective metacyclic trypomastigotes, and again the phosphorylation of eIF2α T169 correlates with the levels of overall translation Trypanosomes separated early from other eukaryotes and have some divergent features in the structure and regulation of eIF2. First, eIF2α has an amino terminal extension of >100 residues when compared with animals, fungi or higher plants. Second, the phosphorylated residue that aligns with S51 is threonine 169 (T169) in T. brucei it has been shown that phosphorylation of eIF2α T169 is not necessary for either arrest or the formation of stress granules during heat shock T. brucei cell line containing a single eIF2α gene with a T169A mutation was used to determine whether phosphorylation of eIF2α T169 is necessary for either the growth arrest that occurs in stumpy bloodstream forms or the subsequent differentiation to procyclic forms. Both differentiation processes occurred with the same kinetics as the wild type control.In 22.1T. brucei EATRO 1125 bloodstream form cells were grown in HMI-9 medium salts 2.27 cells were harvested and washed twice in serum free HMI-9. The cells were resuspended in 100 μl SDS-PAGE sample buffer and incubated at 100 °C for 3 min. Standard SDS-PAGE and Western blotting procedures were used, rabbit anti-eIF2α was diluted 1:5000 1 × 102.3The plasmid for deletion of the first allele of eIF2α and both plasmids for the substitution of the second allele for either the mutant or the wild type TbeIF2α have been previously described 2.47 cells resuspended in 100 μl of Amaxa Human T-cell buffer with 10 μg of linearized plasmid DNA in 0.2 mm cuvettes and program X-001 of the Amaxa Nucleofactor II (Lonza). Transfectants were selected and cloned in HMI-9 medium supplemented with G418 (2.5 μg/ml) and/or blasticidin (5 μg/ml) as required.Transfections were performed using 3 × 102.5After the antibiotic selection, clones were screened by amplification and sequencing of the region corresponding to the phosphorylation site region (T169). PCR was performed with Expand High Fidelity PCR (Roche) using oligonucleotides D526 (ATGGCAGCTTACGGTATAGTG) and D527 (AACCTCATTTCCCTTGAAGAA); the amplified region covered nucleotides 1–675 of the open reading frame. The PCR products were sequenced without cloning using oligonucleotide D527 as a primer.2.65 cultured bloodstream form trypanosomes. Four mice were used for each of the eIF2A T169A/- and eIF2A T169T/-cell lines. The subsequent parasitaemia was followed daily from day 2, trypanosome density measured using a haemocytometer after diluting 5 μl blood in 45 μl 0.83% ammonium chloride solution and further 10-fold dilutions as necessary.CD1 mice were inoculated intraperitonially with between 5 and 15 × 102.7In vitro differentiation was initiated from blood collected 5 days after infection, as above. All populations were predominantly stumpy in morphology. The blood containing the parasites was incubated in DTM medium with 3 mM citrate/cis-aconitate at 28 °C at a density of 2 × 106 trypanosomes/ml. After 6 h the red blood cells had settled and half the supernatant was removed to a new culture flask and proliferation rate was measured after 24 h.2.8Glossina pallidipes) were fed with bloodstream form or cultured procyclics in washed red blood cells from horse (estimated 8 × 106 parasites/ml), supplemented with 10 mM l-glutathione to increase infection rates 24 to 48 h post-eclosion and as the first feed, experimental tsetse flies  that contained a T169A mutation as previously described eIF2A+/− cell lines. Second, a T169A mutation was introduced into the remaining allele, eIF2A T169A/-. This step altered the 3′ untranslated region of the eIF2A gene and a control cell line was made with the same change in the 3′ untranslated region but with wild type eIF2A open reading frame sequence, eIF2A T169T/-.eIF2A open reading frame from genomic DNA followed by direct sequencing of the PCR product , eIF2A T169T/- and eIF2A T169A/-, were used to infect tsetse flies to investigate these transitions in two different experiments (eIF2A T169T/- and eIF2A T169A/- showed reduced ability to establish salivary gland infection. Taken together, these observations indicate that phosphorylation of eIF2α at T169 is not necessary for the differentiation as far as the proventriculus, including the cell cycle arrest prior to the asymmetric cell division.There are at least three further points in the life cycle that include a cell cycle arrest in G1. One occurs in procyclic cells as they migrate towards and invade the tsetse proventriculus eriments . In the eriments . No infeeriments . However4eIF2A allele expressing a non-phosphorylatable eIF2α T169A was able to enter and exit quiescence in a similar manner to a wild type control. The ability of the mutant cells to enter and leave cell cycle arrest with wild type kinetics provides strong evidence that the phosphorylation of T169 is not sufficient to promote these transitions that are associated with changes in protein synthesis. The only phenotype observed was a slightly slower proliferation rate as bloodstream forms in culture.When a cell switches between quiescence and proliferation both the identity of proteins made and the rate of protein synthesis are altered. In trypanosomes, the non-selective polycistronic transcription of protein coding gene arrays means that there is an apparent conflict between maintaining/increasing the expression of proteins associated with quiescence and decreasing the overall rate of protein synthesis. Here, any role for phosphorylation of eIF2α on T169 in regulating differentiation between proliferative and quiescent developmental forms has been investigated. The main finding is that a cell line with a single T. cruzi and Leishmania infantum. In T. cruzi epimastigotes, the level of phosphorylation of T169 increases under a nutritional stress that results in differentiation to metacyclics, at the same time as the number of polysomes decreases, providing evidence that the overall rate of protein synthesis could be regulated by eIF2α phosphorylation. Overexpression of eIF2α mutated in the phosphorylation site has a dominant effect and reduced differentiation from the proliferative epimastigote to the non-proliferative metacyclic form Leishmania, a slowing in growth associated with differentiation of promastigotes to amastigote forms also coincides with eIF2α phosphorylation and overexpression of an eIF2α kinase deleted for its regulatory domain and sequences necessary for correct localization slowed differentiation T. cruzi and are consistent with a role for eIF2α phosphorylation in the differentiation of Leishmania after the uptake by macrophages and acidification in the parasitophorous vacuole.In kinetoplastids, the evidence for a functional role for eIF2α T169 phosphorylation in regulating the overall rate of protein synthesis comes from T. brucei slender to non-proliferating stumpy forms does not occur as a consequence of nutritional starvation or environmental changes. Similarly, the formation of procyclics in the insect gut does not appear to depend on the lack of nutrients T. brucei and the other studied kinetoplastids, and they represent the first direct evidence indicating that phosphorylation of eIF2α T169 is not necessary for controlling these T. brucei differentiation events In contrast, the differentiation of proliferating T. brucei transformation of epimastigotes to metacyclics in the insect salivary gland, a differentiation process that occurs in an environment that is poor in nutrients.Given the relevance of phosphorylation of eIF2α at T169, or equivalent residue, in differentiation events in other parasites, it is possible that this phosphorylation and consequent inhibition of translation could be required for the


Ascaris lumbricoides specific IgE and eosinophils in children from endemic areas to assess the Th2-type immune response in a population that comprised 205 children with aged 1-10 years, and of low socioeconomic status.This study investigated the relationship between total IgE and A. lumbricoides specific IgE were determinated by ImmunoCAP (Pharmacia) and the eosinophils were counted in peripheral blood.Fecal samples were analysed by the methods of Blagg and Kato-katz. The serum levels of total IgE and A. lumbricoides was detected in 140 (68%) children. The levels of total and specific IgE and eosinophils presented values above those of standard reference . Total IgE, A. lumbricoides specific IgE and eosinophils were significantly higher in the A. lumbricoides positive children as compared to A. lumbricoides negative ones .The results showed a prevalence of 89% (182) for intestinal parasites. A. lumbricoides, induced a Th2-type immune response with production of the total and specific IgE and eosinophils.Our results showed that parasites intestinal infection, particularly


Cores were analysed for texture, salinity, and chl a. We identified annual growth layers for 7 of 11 MYI cores and found no significant differences in chl a concentration between the bottom first-year-ice portions of MYI, upper old-ice portions of MYI, and FYI cores. Overall, the maximum chl a concentrations were observed at the bottom of young FYI. However, there were no significant differences in chl a concentrations between MYI and FYI. This suggests little or no change in algal biomass with a shift from MYI to FYI and that the spatial extent and regional variability of refrozen leads and younger FYI will likely be key factors governing future changes in Arctic sea ice algal biomass. Bottom-integrated chl a concentrations showed negative logistic relationships with snow depth and bulk (snow plus ice) integrated extinction coefficients; indicating a strong influence of snow cover in controlling bottom ice algal biomass. The maximum bottom MYI chl a concentration was observed in a hummock, representing the thickest ice with lowest snow depth of this study. Hence, in this and other studies MYI chl a biomass may be under-estimated due to an under-representation of thick MYI , which typically have a relatively thin snowpack allowing for increased light transmission. Therefore, we suggest the on-going loss of MYI in the Arctic Ocean may have a larger impact on ice–associated production than generally assumed.With near-complete replacement of Arctic multi-year ice (MYI) by first-year ice (FYI) predicted to occur within this century, it remains uncertain how the loss of MYI will impact the abundance and distribution of sea ice associated algae. In this study we compare the chlorophyll  Arctic first-year sea ice (FYI), from lower latitude and shelf regions, is generally more productive than multi-year ice (MYI), which leads to the assumption that a replacement of MYI by FYI will result in an overall increase of sea ice algal biomass. Arctic sea ice has already undergone a dramatic reduction of MYI with pronounced losses of the oldest and thickest MYI –3. In SeThe rapid loss of sea ice represents an equally rapid change in habitat for sea ice algae, protists, and ice-associated fauna. Sea ice algae represent an important and high quality food source, directly or indirectly, for many key organisms found in polar regions . In Sea ice decline, thinning of Arctic sea ice, and the loss of MYI have resulted in reduced Arctic-wide sea ice albedo  and more-1[-1[a (chl a) and snow depth , nutrient supply, and transmitted irradiance (see recent review in ). During-1–31, and -1[-1,32–34. Ih e.g., ,36). Dur. Dur-1[-h , how, howa foI . These a biomass  gaps.A summary of studies concerning Arctic MYI chl  biomass  reveals  biomass . The fou biomass  leaving  biomass , none chThe north-eastern coast of Canada, including the Lincoln Sea, represents an important region as it is home to some of the oldest and thickest ice in the Arctic and will likely be one of the last remaining refuges for MYI in the future ,5,53,54.a biomass than MYI in the Lincoln Sea and discuss the implications of our results in the context of a future Arctic with little or no MYI. We address our scientific question first by providing detailed analyses of the physical properties and chl a concentrations of sea ice (both MYI and FYI) in three consecutive spring seasons, from a region where no similar studies have been reported. Then we evaluate potential differences of ice-algal chl a concentrations and biophysical sea ice properties between ice types, ice ages, and texture classes. Lastly, we investigate the relationship between sea ice chl a concentrations and environmental properties, such as snow depth, sea ice structure, and light availability.The main goal of our study was to determine if FYI has, or has the potential for, higher chl In order to conduct this research project in accordance with regulations set forth by the governing agencies responsible for the study region, all relevant research licenses and permissions were acquired from the Nunavut Research Institute  and Nunavut Impact Review Board (Screening Decision Report 08YN057).The Lincoln Sea  has beenThe Lincoln Sea is a dynamic area due to interaction with, and exchange of, sea ice with the Arctic Ocean. The Lincoln Sea ice cover is comprised of immobile landfast coastal sea ice at the southern edges and mobile pack ice at its northern extent. The landfast ice consists primarily of consolidated pack ice with smaller amounts of FYI forming in the interstitial space during freeze-up. The division between landfast ice and pack ice is not a distinct line but rather a transitional region that can be characterized by ice with limited mobility due to geographic barriers and the intermittent nature of ice export to the south through Nares Strait. Sea ice in the Lincoln Sea typically comes from the Central Arctic Ocean transported by the Beaufort Gyre and Transpolar Drift circulation patterns ,60. HoweSampling was conducted during spring in the first two weeks of May 2010, 2011 and 2012 in the Lincoln Sea. One site was located north of the Lincoln Sea . Sea iceDuring this time of year, when temperatures are typically below -10°C and the low salinity MYI is very hard, coring thicker than ~3.5 m becomes exponentially more difficult. We therefore chose relatively level sites that we knew were below ~3.5 m based on pre-drilled 2 inch auger thickness holes.a (“chl a core”) and one to two cores for microbial genetics  of the texture cores proportionally. All cores were transported from the field back to the Canadian Forces Station (CFS) Alert, Nunavut, Canada  and stored at -15 to -20°C in the dark.At each site three ice cores were extracted within 1 m of each other using a 9 cm inner diameter ice corer (Kovacs Enterprise Mark II) and stored in sterile U-Line bags. One core was sampled for texture and bulk salinity (“Texture core”), one core for chlorophyll ere, see ). Due to2 = 0.94). Brine volume estimates were calculated for cores with temperature measurements using equations in [At each core location, snow depth (here after referred to as core-location-snow-depth), freeboard and core length were measured. These measurements represent the local conditions  at sampling locations, which should not be confused with the larger-scale snow depth and ice thickness survey measurements. Internal ice temperatures were measured on texture cores by drilling holes and inserting a thermometer (Testo 720) immediately after core extraction. Temperatures were measured from surface to bottom at intervals of 0.1 m  and 0.5 m (core 5–10). In 2012, only ice surface temperature (depth 0.1 m) was measured. For these cores the internal ice temperatures were linearly interpolated between the surface and assumed  bottom temperature of -1.78°C with typical surface water salinities in the Lincoln Sea of ~32 . During tions in . Brine vSnow depth and ice thickness surveys were conducted along transects adjacent to each coring site. These measurements represent the larger-scale characteristics of the sampled ice floes, which should not be confused with the point measurements: core-location-snow-depth and core length. Snow depth was measured using a metal probe at 1 or 10 m intervals and ice thickness was measured in 2 inch augers holes drilled in the ice at 10 m intervals. The length of each transect and number of measurements were dependent on ice type and time constraints . Snow density measurements were calculated for 5 snow samples collected, at site 2–10, using an Adirondack snow sampler. Additional density values from the same study region were acquired during the CryoSat Validation Experiment .-2 s-1; data acquired from NOAA / ESRL / GMD / GRAD, the GMD-Radiation Group, ftp://aftp.cmdl.noaa.gov/data/radiation/baseline/alt/).Air temperature data were provided by the Environment Canada weather station located on shore at CFS Alert, Nunavut. Mean daily air temperatures during the study were on average -12.5°C (2010 to 2012 combined), with a range between -20.3 to -7.4°C, and a maximum temperature of -3.1°C. Downwelling total solar irradiance measurements representative of the sampling area were also measured at the nearby CFS Alert weather station. Mean and range of values were calculated for the period May 1–11, 2010 to 2012  from the corresponding texture core was assigned. For surface pieces the uppermost texture class was always assigned to the section because deteriorated ice and snow-ice are distinct layers and only located at the surface.Texture analysis was conducted at ~ -15°C. Texture cores were cut into 0.10 to 0.15 m vertical sections that were further cut into vertical thick sections ~ <5 mm thin, using an electric band saw. For each section, the ice remaining after cutting was put into plastic containers, melted and analyzed for bulk salinity using a salinometer (WTW 3300i). Bulk salinities are reported in parts per thousand (ppt). Thick sections were imaged under crossed polarizers. Analysis of the images provided a stratigraphic description of each ice core by identifying different ice texture classes and the boundaries between classes. Here we divided ice types into 7 texture classes based on grain structure and appearance, following classification systems outlined in –67Table. For eacIn MYI cores, we identified the previous year’s annual layer in 7 cores by identifying peaks in bulk salinity profiles that corresponded with changes in crystal structure at the same depth, as described in ,69. Accoa measurements, these cores were always stored below -15°C in the dark, for a maximum of nine days. The cores were then shipped, via air at a maximum temperature of -10°C in the dark, to Resolute Bay, Nunavut, where they were stored below -20°C in the dark for 1 to 2 days. Cores were cut using an electric band saw (sterilized with 95% ethanol) in a -20°C walk-in freezer. Cores were cut into 10 cm sections except for end pieces (range: 0.09 to 0.17 m), placed in sterile Whirl-Pack (NASCO) bags and melted in the dark. End pieces for 2010 and 2012 samples refer to the ice-air interface. The 2011 cores were cut in the opposite direction, therefore these end pieces refer to the ice-water interface. Core sections were melted without the addition of filtered sea water (FSW) because we were also measuring dissolved constituents  in the core sections. It has been shown that for common biological analyses  melting without the addition of FSW is an acceptable procedure [In order to minimize any potential influences on the chl rocedure .a concentrations were determined on sub-samples filtered onto Whatman GF/F filters, after 24 h extraction in 90% acetone at 4°C in the dark, using a 10AU Tuner Design fluorometer calibrated with pure chlorophyll extract from Anacystis nidulans  or are vertically integrated (mg m-2) for the bottom 0.2 m core sections (hereafter referred to as bottom-integrated), age class core sections, or total core length.Chl  . Chl a ons from  and corra data were highly skewed. To achieve the normal distribution patterns required for parametric statistical analyses, log-transformations were applied to the chl a data. Two-sample t-tests were conducted to determine the effect of ice type (MYI & FYI) on the chl a concentrations of the sea ice. Variance analyses (ANOVAs) were conducted in order to determine the effect of ice age class ; year and texture class on the chl a concentrations of the ice. Post-hoc tests (Tukey’s HSD) were conducted when both parametric ANOVA (transformed data) and non-parametric Kruskal-Wallis (non-transformed data) analyses showed significant differences.Initial data exploration demonstrated that the distributions of chl a concentrations a logistic regression model was applied. A logistic regression was used in order to identify potential critical values (inflection point) of the independent variables  that could indicate a threshold value for optimal algal growth. Logistic regression analysis required values of the dependent variable in the range 0 to 1.Therefore, bottom-integrated chl a values were normalized to the range 0 to 1 by dividing each chl a value (yi) by the maximum value for all cores (ymax). The relationship between bottom-integrated chl a concentrations and bulk integrated extinction coefficients, for visible radiation, was analyzed in a similar manner. For all calculations we used extinction coefficients for snow: ks = 20.0 m-1 [m = 1.55 m-1; and FYI: kf = 1.45 m-1 [s, used here, was chosen from a table of ks values [-3 . The values of ks, km, and kf were integrated over the depth of the corresponding ice and snow layers for each core site resulting in “integrated extinction coefficients” (dimensionless), i.e., a value for each snow and ice layer at each site. The “bulk (snow plus ice) integrated extinction coefficient” is simply the sum of the integrated extinction coefficients for snow and ice , i.e., one value for each core site. In the resulting bulk integrated extinction coefficients, larger values mean shallower penetration of light.To investigate the potential influence of snow depth and sea ice optical properties on bottom-integrated chl 20.0 m-1 ; MYI: km1.45 m-1 . The vals values  based onResults are reported as arithmetic mean ± one standard deviation (μ̂ ± 1σ).All statistical analyses were conducted with the R software package v-2.15.2 .Based on site-averaged drill hole thickness measurements, i.e., characterizing the larger scale ice properties of the sampled floes, MYI sites were more than twice as thick (3.28 ± 0.56 m) as FYI sites (1.42 ± 0.42 m). Mean MYI core length (2.62 ± 0.24 m), i.e., only characterizing local ice properties of core sampling locations, was also nearly twice as thick as FYI (1.39 ± 0.52 m), with higher variability in FYI core lengths. FYI sites represented two different kinds of ice: ice that formed during the fall when the landfast ice consolidated ; and ice that formed later in mobile ice when open leads formed and then refroze .-3 (n = 5) and during CryoVex 2011 was 281 ± 0.7 kg m-3 (n = 11 [Site-averaged snow depth at MYI sites (0.39 ± 0.10 m), in general, was thicker than at FYI sites (0.26 ± 0.15 m). Although MYI had lower variability between site-averaged snow depth values, FYI had lower variability when considering each site individually. This was illustrated by the mean of site standard deviations for FYI snow depth: 0.08 m, compared to MYI: 0.17 m. Mean snow density at site 2–10  was 260  (n = 11 ).2 = 0.56, p = 0.05, n = 7), and between snow depth and mean ice thickness survey measurements . For MYI, there was an inverse relationship between snow depth and ice core length , and no significant relationship between snow depth and mean ice thickness survey measurements .There was no significant inter-annual difference in the mean physical properties (e.g. snow depth and ice thickness) of FYI or MYI . For FYI, there were significant positive relationships between core-location-snow-depth and ice core length . All FYI cores followed typical C-shaped bulk salinity curves , except Based on the measured length of each texture class, FYI cores consisted predominantly of columnar ice (76%), with only minor proportions of granular (17%) and mixed (7%) ice. Retextured, melt pond, snow-ice and deteriorated texture classes were not identified in FYI and represented ≤5% of MYI but in some instances represented up to 50% of MYI : core 3–10 (14.1 mg m-3) and 4–11 , which corresponded to a MYI hummock with the lowest core-location-snow-depth, and core 1–12 , which had a brine volume peak at the same depth as the chl a peak. In all MYI cores with confirmed annual layers, peaks in chl a concentrations closely matched the depths of the annual layers  than FYI (0.71 ± 0.92 mg m-2). These differences, however were not statistically significant . We also found no significant effect of ice type on volumetric or areal chl a concentrations  in the bottom 0.2 m were significantly higher in FYI than in MYI  on the chl trations .a concentrations were significantly higher in landfast ice than in pack ice  was representative of the region when compared to the mean snow depth of 0.3 m found by . The conThe FY portions of the MYI, and all of the FYI cores were representative of ice forming locally in the Lincoln Sea during the previous winter. FY ice portions had thicknesses between 0.4 and 0.7 m, which agrees well with reported literature values for new-ice growth at the bottom of MYI between 0.45 and 0.55 m ,73,75. Ba peaks were observed to coincide with all of the identified annual layers and are probably remnants from the previous year’s algal communities. A previous study [a peaks that corresponded with a transition from mixed to columnar ice types, but did not conclude it was an annual layer. The correspondence of both texture and bulk salinity profiles with local chl a peaks and the agreement with literature values provides strong evidence that the internal chl a peaks corresponded to the bottom ice algae layers from previous years.Annual layers in MYI cores were identified at the transition from columnar ice texture of FY portions to granular and mixed texture in the MY portion. Local chl us study  also obsa concentration (1.9 mg chl a m-2) with maximum bottom-integrated chl a concentrations reported for different regions during spring , and in the Baffin Bay and the Beaufort Sea they were over one order of magnitude higher (24 to 64 mg chl a m-2). The large majority of these and other sea ice studies, however, focus primarily on FYI [a concentrations is limited to only a few studies. Our range of core-averaged MYI chl a concentrations (0.1 to 0.8 mg chl a m-3) is in agreement with other spring MYI values , however, was an order of magnitude higher than maximum FYI core-averaged concentrations measured during that study in April to May (~0.2 mg chl a m-3). Melnikov et al. (2002) [a concentrations during July providing some evidence that our study was conducted during the early stages of the growth season, and maximum biomass levels had not yet been reached. Spring MYI chl a concentration values comparable to our study were also reported from the Bering Sea with core-integrated concentrations between 0.3 to 3.0 mg chl a m-2 [a m-2 [a m-3 [A comparison of our maximum bottom-integrated chl mmary in  and refey on FYI . Consequhl a m-3 ), observ. (2002)  reportedhl a m-2 , the Gre2 [a m-2 , and Fra2 [a m-3 .a biomass are observed in FYI at the same location due to small scale variations in snow and ice properties , regardless of large differences in their physical properties.It is well documented that differences in chl eview in ). We hava concentrations than the other ice age portions, because it experienced vertical flushing of the ice column during the previous melt season(s), and had no access to nutrient replenishment from the underlying water column. However, during our study the upper MY still had chl a concentration values similar to FY and FYI. This observation can be attributed to the presence of remnant communities within the previous year’s bottom layers and melt ponds. Refrozen melt-ponds were identified at or near the surface of 6 out of 11 MYI cores. Each coincided with an elevated chl a concentration in the corresponding core section. Algal communities in surface layers of Arctic sea ice are not common. However, similar features were observed in summer sea ice by Gradinger et al., (2005), and were attributed to freshwater melt pond inhabitants [It might also be expected that the upper MY portion would have had lower chl abitants .a maxima, observed throughout the core, originate from phototrophic eukaryotes (i.e. diatoms and flagellates), which is in agreement with a previous study that reported high flagellate and diatom biomass in the upper and bottom portions, respectively, of Arctic summer ice [Maintenance of the previous years’ algal biomass levels in the annual layers was supported by a separation of bacterial communities in one MYI core. At MYI site 1–11, distinct bacterial assemblages were observed at different depths  based on analyses of the 16S rRNA gene from one coincident core . This summer ice .a concentrations at sites with the lowest snow depth and highest potential light availability indicates that the limiting factor for algal growth during this study was light availability. This is consistent with other studies that found light to be limiting algal growth during the early-spring growth season . The. Thea coa concentrations were nearly zero at snow depths >0.17 m, or at bulk integrated extinction coefficients >5.8 ; or 2) not favorable for algal growth (lower chl a).The logistic regression analysis showed that core-normalized bottom integrated chl >5.8 Fig  and 8c. h on FYI . The mai-2 s-1. This estimate is in good agreement with reported critical minimum under-ice irradiance levels to maintain algal growth between ~2 and 9 μmol photons m-2 s-1 [a concentration emphasizes the dominating effect of snow depth on light transmission and subsequent algal growth. The combined effect of snow and ice on light transmission and subsequently on chl a concentration in the lowermost 0.2 m of sea ice demonstrates that both parameters should be considered when comparing ice of variable thicknesses and snow depths.Combining the mean downwelling incoming radiation values with the critical threshold bulk integrated extinction coefficient value (5.8) as parameters in a simplified light extinction model (equation 1 in ), result m-2 s-1 –85. The -1 . This suggests that light limitation and self-shading by in-ice algae would have been minimal during our study.Although snow typically has a dominating effect on light transmission compared to sea ice, which has typical extinction coefficients between 0.8 and 1.55 m1 e.g., ,32–34), –34, -1 . The. The-1  that was also from a younger refrozen lead did not have high biomass even though it was the thinnest core and had lowest snow depth. This could be explained by higher under-ice irradiance, which would have inhibited algal colonization until light levels became more favorable or until algal cells would have had sufficient time to adapt to the light conditions  in the Lincoln Sea and in a future Arctic system with a more mobile ice pack the relative coverage of refrozen leads will likely increase and perhaps become an increasingly more important component of overall sea ice algal biomass in the Lincoln Sea and Arctic Ocean [A common feature, which influences the formation of refrozen leads and FYI, in the Lincoln Sea is an ice arch that forms at the entrance to Nares Strait ,90. Withic Ocean .a concentration was extracted from a hummock. The hummock core, 5–10, corresponded to the thickest core with shallowest snow depth and high chl a biomass . Light attenuation is likely different between refrozen melt pond ice and hummocks, which is apparent when you compare the texture images for deteriorated ice . ,91. a coIn light of the limited number of recent studies, one may argue that our current understanding of Arctic sea ice algal biomass in MYI is based on a historic Arctic that was different from today. The melt season has lengthened ,7, and sa biomass in FYI compared to MYI. FYI values include highly productive regions (e.g. Arctic shelves) where MYI is not present or has not been studied. Here we show that FYI and MYI in the Lincoln Sea can have comparable chl a biomass during the spring period (May). Previous studies have also demonstrated comparable or even slightly higher chl a biomass in MYI compared to FYI [a biomass values have also been observed in the thickest sea ice during spring [a biomass potential in MYI compared to FYI, we suggest that the general view of higher productivity in FYI than in MYI should be revisited in order to achieve a better understanding of the current and future state of the Arctic system.Arctic FYI studies in general have shown a higher range of bottom chl d to FYI . In addig spring  and summg spring . Based oa biomass potential of thick MYI (hummocks) suggests the on-going loss of MYI in the Arctic Ocean may have a larger impact on ice–associated production than generally assumed.If we base future estimates of ice-algal production on the fact that FYI from Arctic shelf regions is more productive than MYI in general, this would lead to the assumption that sea ice algal production would increase with a replacement of MYI by FYI during the ice-covered period. However, our results suggest only minor changes in ice algal biomass when all MYI is replaced by FYI. This considered in combination with the underestimated chl a biomass and that the comparable values between MYI and FYI are likely driven by the higher biomass in the younger FYI. The relative proportion of younger FYI and refrozen leads in the central Arctic Ocean will likely increase with continued increases in ice drift velocities and a thinning ice pack [Our results also showed that younger FYI  had the highest chl ice pack ,13,93. TThe expected higher bloom biomass of thinner FYI, however, may not result in a net increase in ice algal production over the entire growth season. Even with larger areas of thinner FYI, the expected increase in maximum ice-algal biomass may not compensate for the increased vulnerability of thinner ice to rapid changes in the light field and rapid snow/ice melt. These vulnerabilities could result in earlier termination of the ice-algal bloom due to photo-inhibition and/or rapid melt ,40. Earla and physical properties of both FYI and MYI during spring from a high-Arctic system. The low variability in chl a concentrations, both within and between MYI and FYI in the coastal Arctic Ocean, suggests little or no change in algal biomass with a shift from MYI to FYI. The apparent relationship between chl a biomass in the bottom layer of ice and bulk integrated extinction coefficients of the snow-ice matrix, implies that an appropriate representation of areas with low snow depths, such as MYI hummocks, is critical for a realistic estimation of the MYI contribution to overall ice algal biomass estimates in the Arctic Ocean. The potential for higher ice algal biomass in thick MYI with less snow, in conjunction with a lack of significant difference between FYI and MYI chl a biomass during our study suggests that the on-going loss of MYI in the Arctic Ocean may have a more negative impact on ice–associated production than generally assumed.Studies comparing biogeochemical properties of first-year sea ice (FYI) with multi-year sea ice (MYI) in the high Arctic are essential to understand the potential biogeochemical changes to sea ice ecosystems in a future Arctic Ocean with little or no MYI. In light of the current limited investigations of Arctic MYI algae, the present study provides a unique multi-annual dataset comparing ice-algal chl


The aim of this study was to use aqueous suspensions of silver nanoparticles with a wide spectrum of particle sizes, variable morphology, high stability, and appropriate physicochemical properties to examine their bactericidal and fungicidal properties against microorganisms present in poultry processing plants. At the same time, the particles were tested for preventing the production of odorogenous pollutants during incubation and thereby reducing the emission of harmful gases from such types of facilities. The results show that the use of nanosilver preparations in order to disinfect eggs and hatchers reduced microbiological contamination. The bactericidal and fungicidal efficacy of the applied preparation was comparable to UV radiation and its effectiveness increasing during the incubation. Good results were achieved in terms of the level of organic gaseous contaminants, which decreased by 86% after the application of the nanosilver preparation. Poultry farming and related chick hatching processes, which base their production technology on biological material, are a potential source of microbiological contamination, including microorganisms and their toxins. These factors are one of the components of bioaerosols, particularly organic dust. They are shunted outside through a ventilation system and remain at high levels up to one kilometre from the source of emission. Many poultry diseases, including respiratory tract infections, are transmitted through the air over long distances, even reaching locations three kilometres away from the place of formation –8.3 and increased up to 310,000 cfu/m3 when the chicks were being taken out. Nowadays, even the new technologies introduced to modernised plants according to the requirements of the European Union do not totally eliminate biological air pollution. Research by Tymczyna et al. . The following formulas were used in the calculations:(i)N):Test bacteria suspension The number of viable bacteria in (Na): c is the sum of colonies counted on both plates, n is the number of plates included in the calculation, d is dilution factor, and V is sample volume.where (iii)X):Degree of reduction of viable bacteria  consisted of hen eggs disinfected with nanosilver before putting them in the incubator (Incubator D). The control group (Group K) consisted of eggs disinfected using UV irradiation for 30 minutes before putting them in the incubator (Incubator K). In the experimental group, the hatcher and the eggs were fogged with a nanosilver preparation. In both groups, the disinfection efficacy was evaluated based on microbiological analyses of the egg surfaces and the hatchers.The analyses were performed before (series I) and 30 minutes after disinfection (series II). During the incubation, experiments were carried out three times, that is, after the 1st (series III) and 7th day, on the day of candling the eggs (series IV), and on the 17th day, before the onset of hatch (series V). Staphylococcus count, and total fungal count.The microbiological analysis of the egg surfaces and the hatchers is comprised of the determination of the total bacteria count, total3 of sterile distilled water with Tween 80. After 15 minutes of shaking, cultures were performed from the obtained washings by the surface plating technique on proper media . After collection, smears were placed in transport tubes containing 20.0 cm3 of saline. The collected samples were shaken for 2 minutes. From the obtained suspension, cultures were performed on proper media  and control apparatus (UV Incubator) before and 30 minutes after disinfection. During the incubation, the experiments were carried out three times, that is, after the 1st (series III) and 7th day, on the day of candling the eggs (series IV), and on the 17th day, before the onset of hatch (series V). The concentrations of volatile organic compounds (VOCs) and inorganic compounds, particularly sulphur compounds with strong odorogenous properties, were determined in the collected air samples.Organic gaseous air contaminants were determined by gas chromatography. Air samples were collected with an electric pump into 2-3-litre Tedlar bags . The organic compounds contained in the air samples were condensed and then desorbed using a thermal desorption unit  to the gas chromatography system  equipped with a selective flame photometric detector (FPD) combined with an S-filter with a 393 nm wavelength.Determination of the inorganic compound content in samples sucked into bubbling washers was performed by ion chromatography with the use of a liquid chromatograph (Waters) connected to an analytical column  associated with a conductivity detector and UV Indicator.On the basis of microbiological analyses  conducte Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus than preparations 6, 7, and 8. This could be the result of the positive electrokinetic potential and thus a weaker influence on cell membranes of the tested microorganisms, as well as strong steric stabilisation achieved due to gelatine application and, as a consequence, restricted possibility of the direct action of silver particles. Gelatine is a protein and, at elevated temperatures, its collagen bonds break. A colloidal system then forms, where the stabilisation of metallic particles is facilitated. At that time, spherical stabilisation occurs; that is, the protein chains, due to their complex structure, are an effective spatial factor which prevents the agglomeration of metal particles by surface adsorption.In the other preparations, a similar degree of reduction was reached only after prolonging the contact time to 30 minutes . Prepara S. aureus was the most resistant bacteria to all of the tested preparations among the tested microorganisms, whereas the yeast C. albicans turned out to be the most sensitive.It was found that the preparations containing silver particles of about 50 nm in size, which were stabilised with gelatine (preparations 2 and 3), showed the weakest antimicrobial effect.5-fold reduction in viable bacteria and 104-fold or greater reduction in the case of fungi, under specific test conditions, in 60 minutes or less. Based on the presented results, it can be concluded that all the preparations achieved the required level of reduction for the majority of the studied strains after only 60 minutes of contact. After 60 minutes of contact, the studied preparations achieved the 105-fold result of reduction for the majority of strains. This situation did not only refer to the Gram-positive bacterium S. aureus which, in the case of the evaluation using the test tube method, proved to be the most resistant to the action of silver nanoparticles. Suspension number 6 achieved the required level of reduction after only 15 minutes in case of the yeast C. albicans  showed disinfecting action in this period of time only towards S. enteritidis. On the basis of these results, preparation number 8 was selected for application tests.The results of the evaluation of the bactericidal and fungicidal properties of nanosilver preparations 6, 7, and 8, which was conducted using the dilution-neutralisation method (PN-EN 1040 and PN-EN 1275), are summarised in Tables albicans . A similalbicans , but thealbicans . After 34 cfu/egg, while the average fungal count was 2.1 × 102 cfu/egg. Therefore, maintaining microbiological purity is possible with properly conducted disinfection of eggs and hatchers. In these tests, the effectiveness of the studied nanosilver preparation in the case of the total bacteria count was on a level similar to the effectiveness of UV disinfection  and cyclobutanol (247.1 μg/m3), whereas, in the NANO Incubator, the highest concentration was seen for hexanal (255.4 μg/m3). In the apparatus disinfected with nanosilver, the presence of 2-pentanamine, 2-methylpentane, 2-methyl-1-propanol, trichloroethylene, and toluene (Among the identified VOC compounds, the highest concentration in the UV Incubator was seen for 2-pentanamine (284.3  toluene  as well  toluene  was not  toluene .According to current standards of production, in the majority of food industry plants, including the egg-poultry industry, the obligatory control of microbiological and chemical quality of air and production surfaces has been introduced, which means an obligation to ensure proper cleanliness. In chick hatcheries, the hatchers constitute a critical point because thermal and humidity conditions as well as the presence of biological material inside them pose a significant microbiological hazard. In such places, not without reason, great emphasis has been placed on maintaining microbiological purity, which is achieved only by properly conducted disinfection of hatchers and eggs.The present research demonstrated that the nanosilver preparation applied for the disinfection of eggs and hatchers reduced microbiological contamination. The preparation used showed bactericidal and fungicidal effectiveness comparable to UV radiation, and its effectiveness increased throughout the incubation. In the case of fungi, on the 7th day of incubation, fogging with nanosilver gave better protection to the surface of eggs than irradiation with UV.Very good results were achieved in the case of organic gaseous contaminants. After application of the nanosilver preparation, these levels decreased by 86%. The level of contaminants in the air inside the incubator decontaminated with UV was 40% higher than in the incubator disinfected with nanosilver.Proper egg incubation conditions during hatching are very important and result in effective hatching and healthy chicks. Intensively running metabolic processes and embryo respiration require that the air inside incubator contains a sufficient amount of oxygen. Gases released during the incubation have to be removed from the apparatus, because excessive amounts can lead to poisoning and embryo decay, so proper air exchange is of great importance. The application of nanosilver may be used to optimise the process of incubation. As this study shows, nanosilver is not able to replace efficiently functioning ventilation but may be an element supporting the elimination of gaseous contaminants.