Heat induced HSP20 phosphorylation without increased cyclic nucleotide levels in swine carotid media

Background  

Heat pretreatment of swine carotid artery has been shown to increase ser¹⁶-heat shock protein 20 (HSP20) phosphorylation and suppress force, i.e., reduce force with only minimal reduction in ser¹⁹-myosin regulatory light chain (MRLC) phosphorylation.     

UreB immunodominant epitope-specific CD8+ T-cell responses were beneficial in reducing gastric symptoms in Helicobacter pylori-infected individuals

Background and aims

Although Helicobacter pylori is recognized as an extracellular infection bacterium, it can lead to an increase in the number of CD8⁺ T cells after infection. At present, the characteristics of H. pylori antigen-specific CD8⁺ T cells and the epitope response have not been elucidated. This study was focused on putative protective antigen UreB to detect specific CD8⁺ T-cell responses in vitro and screen for predominant response epitopes.

Methods

The PBMCs collected from H. pylori-infected individuals were stimulated by UreB peptide pools in vitro to identify the immunodominant CD8⁺ T-cell epitopes. Furthermore, their HLA restriction characteristics were detected accordingly by NGS. Finally, the relationship between immunodominant responses and appearance of gastric symptoms after H. pylori infection was conducted.

Results

UreB-specific CD8⁺ T-cell responses were detected in H. pylori-infected individuals. Three of UreB dominant epitopes (A-2 (UreB_443–451: GVKPNMIIK), B-4 (UreB_420–428: SEYVGSVEV), and C-1 (UreB_5–13: SRKEYVSMY)) were firstly identified and mainly presented by HLA-A*1101, HLA-B*4001 and HLA-C*0702 alleles, respectively. C-1 responses were mostly occurred in H. pylori-infected subjects without gastric symptoms and may alleviate the degree of gastric inflammation.

Conclusions

The UreB dominant epitope-specific CD8⁺ T-cell response was closely related to the gastric symptoms after H. pylori infection, and the C-1 (UreB_5-13) dominant peptides may be protective epitopes.     

Comparative performance of contact plates,electrostatic wipes,swabs and a novel sampling device for the detection of Staphylococcus aureus on environmental surfaces

Aims

To evaluate the performance of four sampling methods [contact plates, electrostatic wipes (wipe), swabs and a novel roller sampler] for recovery of Staphylococcus aureus from a stainless steel surface.

Methods and Results

Stainless steel test plates were inoculated with Staph. aureus, dried for 24 h and sampled using each of the four methods. Samples were either incubated directly (roller, contact plate) or processed using elution and membrane filtration (swab, wipe). Performance was assessed by calculating the apparent sampling efficiency (ASE), analytical sensitivity (Sn) and percentage of replications with positive growth. The wipe demonstrated the best performance across all inoculating concentrations (ASE_48 h = 18%; Sn_48 h = 7 CFU per 100 cm²). The swab performed well when corrected for area actually sampled (ASE_48 h = 24%; Sn_48 h = 76 CFU per 100 cm²). Of the contact‐based methods, the newly developed roller sampler outperformed the contact plate (roller: ASE_48 h = 10%; Sn_48 h = 17 CFU per 100 cm²; contact plate: ASE_48 h = 0·04%; Sn_48 h = 1412 CFU per 100 cm²); both contact samplers performed better at higher inoculating concentrations (6E3 CFU per 100 cm² for the roller and 6E6 CFU per 100 cm² for the contact plate). Overall, the electrostatic wipe produced the highest number of replications resulting in positive growth (74%_24 h, 91%_48 h).

Conclusions

This study demonstrates that selection of the sampling method must be carefully considered, given that different methods have varying performance.

Significance and Impact of the Study

This is the first study assessing static wipes for sampling and one that uses a more real‐world‐relevant 24‐h drying time. The results help with infection control, and environmental health professionals choose better sampling methodologies.     

Expression of novel carotenoid biosynthesis genes from Enterococcus gilvus improves the multistress tolerance of Lactococcus lactis

Aims

To determine whether the carotenoid production improves stress tolerance of lactic acid bacteria, the cloned enterococcal carotenoid biosynthesis genes were expressed in Lactococcus lactis ssp. cremoris MG1363, and the survival rate of carotenoid‐producing engineered MG1363 strain under stress condition was investigated.

Methods and Results

We cloned carotenoid biosynthesis genes from yellow‐pigmented Enterococcus gilvus. The cloned genes consisted of crtN and crtM and its promoter region were inserted into the shuttle vector pRH100, and the resulting plasmid was named pRC. The cloned crtNM was expressed using pRC in noncarotenoid‐producing L. lactis ssp. cremoris MG1363. The expression of crtNM led to the production of C₃₀ carotenoid 4,4′‐diaponeurosporene. After exposure to 32 mmol l^?1 H₂O₂, low pH (1.5, acidified with HCl), 20% bile acid and 12 mg ml^?1 lysozyme, the survival rates of the MG1363 strain harbouring pRC were 18.7‐, 6.8‐, 8.8‐ and 4.4‐fold higher, respectively, than those of MG1363 strain harbouring the empty vector pRH100.

Conclusions

The expression of carotenoid biosynthesis genes from Ent. gilvus improves the multistress tolerance of L. lactis.

Significance and Impact of the study

First report of the improvement of multistress tolerance of lactic acid bacteria by the introduction of genes for carotenoid production.     

Characterization and flocculation properties of biopolymeric flocculant (glycosaminoglycan) produced by Cellulomonas sp. Okoh

Aims

Bioflocculant production potential of an actinobacteria isolated from a freshwater environment was evaluated and the bioflocculant characterized.

Methods and Results

16S rDNA nucleotide sequence and BLAST analysis was used to identify the actinobacteria and fermentation conditions, and nutritional requirements were evaluated for optimal bioflocculant production. Chemical analyses, FTIR, 1H NMR spectrometry and SEM imaging of the purified bioflocculant were carried out. The 16S rDNA nucleotide sequences showed 93% similarities to three Cellulomonas species (strain 794, Cellulomonas flavigena DSM 20109 and Cellulomonas flavigena NCIMB 8073), and the sequences was deposited in GenBank as Cellulomonas sp. Okoh (accession number HQ537132 ). Bioflocculant was optimally produced at an initial pH 7, incubation temperature 30°C, agitation speed of 160 rpm and an inoculum size of 2% (vol/vol) of cell density 1·5 × 10⁸ cfu ml^?1. Glucose (88·09% flocculating activity; yield: 4·04 ± 0·33 g l^?1), (NH₄)₂NO₃ (82·74% flocculating activity; yield: 4·47 ± 0·55 g l^?1) and MgCl₂ (90·40% flocculating activity; yield: 4·41 g l^?1) were the preferred nutritional source. Bioflocculant chemical analyses showed carbohydrate, protein and uronic acids in the proportion of 28·9, 19·3 and 18·7% in CPB and 31·4, 18·7 and 32·1% in PPB, respectively. FTIR and 1H NMR indicated the presence of carboxyl, hydroxyl and amino groups amongst others typical of glycosaminoglycan. SEM imaging revealed horizontal pleats of membranous sheets closely packed.

Conclusion

Cellulomonas sp. produces bioflocculant predominantly composed of glycosaminoglycan polysaccharides with high flocculation activity.

Significance and Impact of the Study

High flocculation activity suggests suitability for industrial applications; hence, it may serve to replace the hazardous flocculant used in water treatment.     

Characterization of polyhydroxyalkanoates accumulated by a moderately halophilic salt pan isolate Bacillus megaterium strain H16

Aim

Characterization of polyhydroxyalkanoates (PHA) accumulated by halophilic bacteria isolated from solar salterns.

Methods and Results

Twenty‐six halophilic isolates were obtained from solar salterns of Goa, India. They were screened for accumulation of PHA by Sudan black B, Nile blue A and Nile red stains. Strains H15, H16 and H26 were selected based on their intensity of Nile blue A/Nile red fluorescence. On the basis of phenotypic and genotypic characterization, the three isolates were identified as Bacillus megaterium. Growth kinetics and polymer accumulating capacity of strain H16 were studied in E2 mineral media with 2% glucose with/without NaCl. In the absence of NaCl, strain H16 accumulated PHA to 40·0% (w/w) of cell dry weight (CDW) at 42 h of growth, whereas in presence of 5% w/v NaCl, the culture showed longer lag phase of up to 24 h and accumulated a maximum PHA of 39% (w/w) CDW at 54 h of growth. The infrared spectra of both the polymers exhibited peaks at 1733·9 cm^?1 characteristic of C=O. Scans of ¹H nuclear magnetic resonance (NMR) showed a doublet at 2·5 ppm corresponding to methylene group (‐CH₂), the signal at 5·3 ppm corresponded to methine group (‐CH‐), and another signal at 1·3 ppm corresponded to the methyl group (‐CH₃). Scans of ¹³C NMR showed prominent peaks at 20, 40, 67–68 and 170 ppm, indicating the polymer to be homopolymer of 3‐hydroxybutyrates. The polymer is stable up to a temperature of 160°C.

Conclusion

Three moderately halophilic isolates (strain H15, H16 and H26) capable of accumulating PHA were isolated from solar salterns of Ribandar Goa, India, and identified as B. megaterium based on phenotypic and genotypic characterization. Strain H16 accumulated polyhydroxybutyrate in the presence and absence of NaCl up to 40% of its CDW.

Significance and Impact of the Study

This strain would be better suited for production of PHA at industrial level due to its tolerance to high concentration of NaCl.     

Gibberellins in dark- and red-light-grown shoots of dwarf and tall cultivars of Pisum sativum: The quantification,metabolism and biological activity of gibberellins in Progress no. 9 and Alaska

The stem growth in darkness or in continuous red light of two pea cultivars, Alaska (Le Le, tall) and Progress No. 9 (le le, dwarf), was measured for 13 d. The lengths of the first three internodes in dark-grown seedlings of the two cultivars were similar, substantiating previous literature reports that Progress No. 9 has a tall phenotype in the dark. The biological activity of gibberellin A₂₀ (GA₂₀), which is normally inactive in le le geno-types, was compared in darkness and in red light. Alaska seedlings, regardless of growing conditions, responded to GA₂₀. Dark-grown seedlings of Progress No. 9 also responded to GA₂₀, although red-light-grown seedlings did not. Gibberellin A₁ was active in both cultivars, in both darkness and red light. The metabolism of [¹³C³H]GA₂₀ has also been studied. In dark-grown shoots of Alaska and Progress No. 9 [¹³C³H]GA₂₀ is converted to [¹³C³H]GA₁, [¹³C³H]GA₈, [¹³C]GA₂₉, its 2-epimer, and [¹³C³H]GA₂₉-catabolite. [¹³C³H] Gibberellin A₁ was a minor product which appeared to be rapidly turned over, so that in some feeds only its metabolite, [¹³C³H]GA₈, was detected. However results do indicate that the tall growth habit of Progress No. 9 in the dark, and its ability to respond to GA₂₀ in the dark may be related to its capacity to 3-hydroxylate GA₂₀ to give GA₁. In red light the overall metabolism of [¹³C³H]GA₂₀ was reduced in both cultivars. There is some evidence that 3-hydroxylation of [¹³C³H]GA₂₀ can occur in red light-grown Alaska seedlings, but no 3-hydroxylated metabolites of [¹³C³H]GA₂₀ were observed in red light-grown Progress. Thus the dwarf habit of Progress No. 9 in red light and its inability to respond to GA₂₀ may be related, as in other dwarf genotypes, to its inability to 3-hydroxylate GA₂₀ to GA₁. However identification and quantification of native GAs in both cultivars showed that red-light-grown Progress does contain native GA₁. Thus the inability of red light-grown Progress No. 9 seedlings to respond to, and to 3-hydroxylate, applied GA₂₀ may be due to an effect of red light on uptake and compartmentation of GAs.Abbreviations AMO-1618 2-isopropyl-4-(trimethylammonium chloride)-5-methylphenyl piperidine-1-carboxylate - cv. cultivar - GC-MS gas chromatography-mass spectrometry - GA_(n) gibberellin A_(n) - HPLC high-pressure liquid chromatography     

[HCO<Subscript>3</Subscript><Superscript>-</Superscript>]-regulated expression and activity of soluble adenylyl cyclase in corneal endothelial and Calu-3 cells

Background  

Bicarbonate activated Soluble Adenylyl Cyclase (sAC) is a unique cytoplasmic and nuclear signaling mechanism for the generation of cAMP. HCO₃ ^- activates sAC in bovine corneal endothelial cells (BCECs), increasing [cAMP] and stimulating PKA, leading to phosphorylation of the cystic fibrosis transmembrane-conductance regulator (CFTR) and increased apical Cl^- permeability. Here, we examined whether HCO₃ ^- may also regulate the expression of sAC and thereby affect the production of cAMP upon activation by HCO₃ ^- and the stimulation of CFTR in BCECs.     

Denitrification in human dental plaque

Background  

Microbial denitrification is not considered important in human-associated microbial communities. Accordingly, metabolic investigations of the microbial biofilm communities of human dental plaque have focused on aerobic respiration and acid fermentation of carbohydrates, even though it is known that the oral habitat is constantly exposed to nitrate (NO₃ ^-) concentrations in the millimolar range and that dental plaque houses bacteria that can reduce this NO₃ ^- to nitrite (NO₂ ^-).     

CD44 expression positively correlates with Foxp3 expression and suppressive function of CD4+ Treg cells

Background  

CD4⁺CD25⁺ regulatory T (T_reg) cells develop in the thymus and can suppress T cell proliferation, modulated by Foxp3 and cytokines; however, the relevance of CD44 in T_reg cell development is less clear. To address this issue, we analyzed Foxp3 expression in CD44⁺ T_reg cells by using multiple parameters, measured the levels of the immunoregulatory cytokine interleukin (IL)-10 in various thymocyte subsets, and determined the suppressor activity in different splenic T_reg cell populations.     

Intracellular calcium changes induced by the endozepine triakontatetraneuropeptide in human polymorphonuclear leukocytes: role of protein kinase C and effect of calcium channel blockers

Background  

The endozepine triakontatetraneuropeptide (TTN) induces intracellular calcium ([Ca⁺⁺]_i) changes followed by activation in human polymorphonuclear leukocytes (PMNs). The present study was undertaken to investigate the role of protein kinase (PK) C in the modulation of the response to TTN by human PMNs, and to examine the pharmacology of TTN-induced Ca⁺⁺ entry through the plasma membrane of these cells.     

CO2 efflux from cleared mangrove peat

Background

CO₂ emissions from cleared mangrove areas may be substantial, increasing the costs of continued losses of these ecosystems, particularly in mangroves that have highly organic soils.

Methodology/Principal Findings

We measured CO₂ efflux from mangrove soils that had been cleared for up to 20 years on the islands of Twin Cays, Belize. We also disturbed these cleared peat soils to assess what disturbance of soils after clearing may have on CO₂ efflux. CO₂ efflux from soils declines from time of clearing from ∼10 600 tonnes km⁻² year⁻¹ in the first year to 3000 tonnes km² year⁻¹ after 20 years since clearing. Disturbing peat leads to short term increases in CO₂ efflux (27 umol m⁻² s⁻¹), but this had returned to baseline levels within 2 days.

Conclusions/Significance

Deforesting mangroves that grow on peat soils results in CO₂ emissions that are comparable to rates estimated for peat collapse in other tropical ecosystems. Preventing deforestation presents an opportunity for countries to benefit from carbon payments for preservation of threatened carbon stocks.     

Effects of stocking density during live transportation on haematological parameters of Siberian sturgeon (Acipenser baerii,Brandt, 1869)

In this study, we investigated the effects of different stocking densities water quality and blood parameters during the transportation of the Siberian sturgeon (Acipenser baerii). Experiments were carried out in high-density polyethylene tank with three different stocking densities (50, 100 and 150 kg/m³) for 20 h. Ammonium nitrogen (NH₄-N) and nitrite nitrogen (NO₂-N) were measured in the water samples. The erythrocyte, leucocyte, haematocrit (HCT), haemoglobin (Hb), cortisol, and sodium ion (Na⁺) were measured in blood samples. Ammonium nitrogen and nitrite nitrogen concentrations in the water have increased in parallel with the stocking density depending on the time. In the highest stocking density, the maximum levels of NH₄-N and NO₂-N at the 16^th h reached 2.64 mg/L and 4.74 mg/L, respectively. Erythrocyte, leucocyte, HCT, and Hb values did not differ significantly between the experimental groups (p > .05). The results showed that fish could be transported safely for 20 h in 15℃ water temperature at 50 kg/m³ stocking density; however, stocking density of 100 kg/m³ and over could threaten fish welfare and health as from 16^th h.     

An automated growth enclosure for metabolic labeling of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> with <Superscript>13</Superscript>C-carbon dioxide - an <Emphasis Type="Italic">in vivo</Emphasis> labeling system for proteomics and metabolomics research

Background  

Labeling whole Arabidopsis (Arabidopsis thaliana) plants to high enrichment with ¹³C for proteomics and metabolomics applications would facilitate experimental approaches not possible by conventional methods. Such a system would use the plant's native capacity for carbon fixation to ubiquitously incorporate ¹³C from ¹³CO₂ gas. Because of the high cost of ¹³CO₂ it is critical that the design conserve the labeled gas.     

Non‐destructive allometric estimates of above‐ground and below‐ground biomass of high‐mountain vegetation in the Andes

Aim

Studies that monitor high‐mountain vegetation, such as paramo grasslands in the Andes, lack non‐destructive biomass estimation methods. We aimed to develop and apply allometric models for above‐ground, below‐ground and total biomass of paramo plants.

Location

The paramo of southern Colombia between 1°09′N and 077°50′W, at 3,400 and 3,700 m a.s.l.

Methods

We established 61 1‐m² plots at random locations, excluding disturbed, inaccessible and peat bog areas. We measured heights and basal diameters of all vascular plants in these plots and classified them into seven growth forms. Near each plot, we sampled the biomass from plants of abundant genera, after having measured their height and basal diameter. Hence, we measured the biomass of 476 plants (allometric set). For each growth form we applied power‐law functions to develop allometric models of biomass against basal diameter, height, height x basal diameter and height × basal area. The best models were selected using AIC_c weights. Using the observed and predicted plant biomass of the allometric set we calculated absolute percentage errors using cross‐validation. The biomass of a plot was estimated by summing the predicted biomass of all plants in a plot. Confidence limits around these sums were calculated by bootstrapping.

Results

For groups of <20 plants the biomass predictions yielded large (>15%) errors. Applying groups that resembled the 1‐m² plots in density and composition, the errors for above‐ground and total biomass estimates were <15%. Across all plots, we obtained an above‐ground, below‐ground and total plot biomass of 329 ± 190, 743 ± 486 and 1011 ± 627 g/m² (mean ± SD), respectively. These values were within the range of biomass estimates obtained destructively in the tropical Andes.

Conclusions

In new applications, if target vegetation samples are similar regarding growth forms and genera to our allometric set, their biomass might be predicted applying our equations, provided they contain at least 50–100 plants. In other situations, we would recommend gathering additional biomass measurements from local plants to evaluate new regression equations.     

Endothelial [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> is an integrating signal for the vascular tone in rat aortae

Background  

Although various endothelium-dependent relaxing factors (endothelial autacoids) are released upon the elevation of endothelial cytosolic free Ca²⁺ concentration (EC [Ca²⁺]_i), the quantitative relationship between EC [Ca²⁺]_i and vascular tone remains to be established. Moreover, whether the basal release of endothelial autacoids is modulated by basal EC [Ca²⁺]_i is still unclear. We assessed these issues by using a novel method that allows simultaneous recording of EC [Ca²⁺]_i and vascular displacement in dissected rat aortic segments.     

Further studies on the metabolism of gibberellins (GAs) A9, A20 and A29 in immature seeds of Pisum sativum cv. progress No. 9

Seed maturation of Pisum sativum cv. Progress No. 9 proceeds more slowly in winter than in summer even when the parent plants are grown in greenhouse conditions with light-and heat-supplementation. For parent plants grown under summer and winter conditions the metabolism of [³H]GA₉ in cultured seeds is qualitatively different in seeds of equivalent age and qualitatively the same in seeds of equivalent weight. 13-Hydroxylation of [³H]GA₉[³H]GA₂₀ is restricted to early stages of seed development. 2-Hydroxylation of [³H]GA₉2-OH-[³H]GA₉ has only been observed at a stage of development after endogenous GA₉ has accumulated. 2-OH-GA₉ has been shown to be endogenous to pea and is named GA₅₁. H₂-GA₃₁ and its conjugate have not been shown to be present in pea and may be induced metabolites of [³H]GA₉. The metabolism of GA₂₀GA₂₉ is used to illustrate a technique of feeding [²H][³H]GAs in order to distinguish a metabolite from the same endogenous compound. The in vitro conversion of [³H]GA₂₀[³H]GA₂₉, and the virtual non-metabolism of [³H]GA₂₉ have been confirmed for seeds in intact fruits. These results are discussed in relation to the apparent absence of conjugated GAs in mature pea seeds.Abbreviations GA_n gibberellin A_n - GC gas chromatography - GC-MS combined gas chromatography-mass spectrometry - GC-RC combined gas chromatography-radio counting - Me methyl ester - R_T etention time - SICM selected ion current monitoring - TLC thin layer chromatography - TMS trimethyl silyl ether The author is née Frydman