Viability Of Strains Research Articles

Clinical and metabolic response to probiotic administration in patients with major depressive disorder: A randomized, double-blind, placebo-controlled trial

ObjectiveWe are aware of no study examining the effects of probiotic supplementation on symptoms of depression, metabolic profiles, serum high-sensitivity C-reactive protein (hs-CRP), and biomarkers of oxidative stress in patients with major depressive disorder (MDD). The present study was designed to determine the effects of probiotic intake on symptoms of depression and metabolic status in patients with MDD. MethodsThis randomized, double-blind, placebo-controlled clinical trial included 40 patients with a diagnosis of MDD based on DSM-IV criteria whose age ranged between 20 and 55 y. Patients were randomly allocated into two groups to receive either probiotic supplements (n = 20) or placebo (n = 20) for 8 wk. Probiotic capsule consisted of three viable and freeze-dried strains: Lactobacillus acidophilus (2 × 109 CFU/g), Lactobacillus casei (2 × 109 CFU/g), and Bifidobacterium bifidum (2 × 109 CFU/g). Fasting blood samples were taken at the beginning and end of the trial to quantify the relevant variables. All participants provided three dietary records (two weekdays and one weekend) and three physical activity records during the intervention. ResultsDietary intake of study participants was not significantly different between the two groups. After 8 wk of intervention, patients who received probiotic supplements had significantly decreased Beck Depression Inventory total scores (−5.7 ± 6.4 vs. −1.5 ± 4.8, P = 0.001) compared with the placebo. In addition, significant decreases in serum insulin levels (−2.3 ± 4.1 vs. 2.6 ± 9.3 μIU/mL, P = 0.03), homeostasis model assessment of insulin resistance (−0.6 ± 1.2 vs. 0.6 ± 2.1, P = 0.03), and serum hs-CRP concentrations (−1138.7 ± 2274.9 vs. 188.4 ± 1455.5 ng/mL, P = 0.03) were observed after the probiotic supplementation compared with the placebo. Additionally, taking probiotics resulted in a significant rise in plasma total glutathione levels (1.8 ± 83.1 vs. −106.8 ± 190.7 μmol/L, P = 0.02) compared with the placebo. We did not find any significant change in fasting plasma glucose, homeostatic model assessment of beta cell function, quantitative insulin sensitivity check index, lipid profiles, and total antioxidant capacity levels. ConclusionsProbiotic administration in patients with MDD for 8 wk had beneficial effects on Beck Depression Inventory, insulin, homeostasis model assessment of insulin resistance, hs-CRP concentrations, and glutathione concentrations, but did not influence fasting plasma glucose, homeostatic model assessment of beta cell function, quantitative insulin sensitivity check index, lipid profiles, and total antioxidant capacity levels.

Antigenotoxic activity of lactic acid bacteria, prebiotics, and products of their fermentation against selected mutagens

Dietary components such as lactic acid bacteria (LAB) and prebiotics can modulate the intestinal microbiota and are thought to be involved in the reduction of colorectal cancer risk. The presented study measured, using the comet assay, the antigenotoxic activity of both probiotic and non-probiotic LAB, as well as some prebiotics and the end-products of their fermentation, against fecal water (FW). The production of short chain fatty acids by the bacteria was quantified using HPLC. Seven out of the ten tested viable strains significantly decreased DNA damage induced by FW. The most effective of them were Lactobacillus mucosae 0988 and Bifidobacterium animalis ssp. lactis Bb-12, leading to a 76% and 80% decrease in genotoxicity, respectively. The end-products of fermentation of seven prebiotics by Lactobacillus casei DN 114-001 exhibited the strongest antigenotoxic activity against FW, with fermented inulin reducing genotoxicity by 75%. Among the tested bacteria, this strain produced the highest amounts of butyrate in the process of prebiotic fermentation, and especially from resistant dextrin (4.09 μM/mL). Fermented resistant dextrin improved DNA repair by 78% in cells pre-treated with 6.8 μM methylnitronitrosoguanidine (MNNG). Fermented inulin induced stronger DNA repair in cells pre-treated with mutagens (FW, 25 μM hydrogen peroxide, or MNNG) than non-fermented inulin, and the efficiency of DNA repair after 120 min of incubation decreased by 71%, 50% and 70%, respectively. The different degrees of genotoxicity inhibition observed for the various combinations of bacteria and prebiotics suggest that this effect may be attributable to carbohydrate type, SCFA yield, and the ratio of the end-products of prebiotic fermentation.

Nasal priming with immunobiotic Lactobacillus rhamnosus modulates inflammation-coagulation interactions and reduces influenza virus-associated pulmonary damage.

To evaluate the effect of the nasal administration of live and heat-killed Lactobacillus rhamnosus CRL1505 (Lr1505) on immune-coagulative response during influenza virus (IFV) infection to improve survival and reduce lung injury. Six-week-old BALB/c mice were treated with live or heat-killed Lr1505 by the nasal route during two consecutive days. Treated and untreated control mice were then nasally challenged with IFV. Both viable and non-viable Lr1505 protected infected mice by reducing pulmonary injury and lung viral loads trough several mechanisms: (a) Inflammatory cytokines were efficiently regulated allowing higher clearance of virus and reduction of inflammatory lung tissue damage, associated to higher levels of the regulatory cytokine IL-10. (b) The antiviral immune response was enhanced with improved levels of type I interferons, CD4(+)IFN-γ(+) lymphocytes, and lung CD11c(+)CD11b(low)CD103(+) and CD11c(+)CD11b(high)CD103(-) dendritic cells. (c) The procoagulant state was reversed mainly by down-regulating tissue factor expression and restoring thrombomodulin levels in lung. The capacity of Lr1505 to improve the outcome of IFV infection would be related to its ability to beneficially modulate lung TLR3-triggered immune response. Our work is the first to demonstrate the ability of an immunobiotic strain to beneficially modulate inflammation-coagulation interactions during IFV infection. Interestingly, non-viable L. rhamnosus CRL1505 was as effective as the viable strain to beneficially modulate respiratory antiviral immune response.

The Kinetic and Thermodynamic Aftermath of Horizontal Gene Transfer Governs Evolutionary Recovery.

Shared host cells can serve as melting pots for viral genomes, giving many phylogenies a web-like appearance due to horizontal gene transfer. However, not all virus families exhibit web-like phylogenies. Microviruses form three distinct clades, represented by φX174, G4, and α3. Here, we investigate protein-based barriers to horizontal gene transfer between clades. We transferred gene G, which encodes a structural protein, between φX174 and G4, and monitored the evolutionary recovery of the resulting chimeras. In both cases, particle assembly was the major barrier after gene transfer. The G4φXG chimera displayed a temperature-sensitive assembly defect that could easily be corrected through single mutations that promote productive assembly. Gene transfer in the other direction was more problematic. The initial φXG4G chimera required an exogenous supply of both the φX174 major spike G and DNA pilot H proteins. Elevated DNA pilot protein levels may be required to compensate for off-pathway reactions that may have become thermodynamically and/or kinetically favored when the foreign spike protein was present. After three targeted genetic selections, the foreign spike protein was productively integrated into the φX174 background. The first adaption involved a global decrease in gene expression. This was followed by modifications affecting key protein-protein interactions that govern assembly. Finally, gene expression was re-elevated. Although the first selection suppresses nonproductive reactions, subsequent selections promote productive assembly and ultimately viability. However, viable chimeric strains exhibited reduced fitness compared with wild-type. This chimera's path to recovery may partially explain how unusual recombinant viruses could persist long enough to naturally emerge.

The Heme Transport Capacity of LHR1 Determines the Extent of Virulence in Leishmania amazonensis.

Leishmania spp. are trypanosomatid parasites that replicate intracellularly in macrophages, causing serious human morbidity and mortality throughout the world. Trypanosomatid protozoa cannot synthesize heme, so must acquire this essential cofactor from their environment. Earlier studies identified LHR1 as a Leishmania amazonensis transmembrane protein that mediates heme uptake. Null mutants of LHR1 are not viable and single knockout strains have reduced virulence, but very little is known about the properties of LHR1 directly associated with heme transport. Here, we use functional assays in Saccharomyces cerevisiae to show that specific tyrosine residues within the first three predicted transmembrane domains of LHR1 are required for efficient heme uptake. These tyrosines are unique to LHR1, consistent with the low similarity between LHR1 and its corresponding homologs in C. elegans and human. Substitution of these tyrosines in LHR1 resulted in varying degrees of heme transport inhibition, phenotypes that closely mirrored the impaired ability of L. amazonensis to replicate as intracellular amastigotes in macrophages and generate cutaneous lesions in mice. Taken together, our results imply that the mechanism for heme transport by LHR1 is distinctive and may have adapted to secure heme, a limiting cofactor, inside the host. Since LHR1 is significantly divergent from the human heme transporter HRG1, our findings lay the groundwork for selective targeting of LHR1 by small molecule antagonists.

Spliceosome assembly in the absence of stable U4/U6 RNA pairing.

The cycle of spliceosome assembly, intron excision, and spliceosome disassembly involves large-scale structural rearrangements of U6 snRNA that are functionally important. U6 enters the splicing pathway bound to the Prp24 protein, which chaperones annealing of U6 to U4 RNA to form a U4/U6 di-snRNP. During catalytic activation of the assembled spliceosome, U4 snRNP is released and U6 is paired to U2 snRNA. Here we show that point mutations in U4 and U6 that decrease U4/U6 base-pairing in vivo are lethal in combination. However, this synthetic phenotype is rescued by a mutation in U6 that alters a U6–Prp24 contact and stabilizes U2/U6. Remarkably, the resulting viable triple mutant strain lacks detectable U4/U6 base-pairing and U4/U6 di-snRNP. Instead, this strain accumulates free U4 snRNP, protein-free U6 RNA, and a novel complex containing U2/U6 di-snRNP. Further mutational analysis indicates that disruption of the U6–Prp24 interaction rather than stabilization of U2/U6 renders stable U4/U6 di-snRNP assembly nonessential. We propose that an essential function of U4/U6 pairing is to displace Prp24 from U6 RNA, and thus a destabilized U6–Prp24 complex renders stable U4/U6 pairing nonessential.

Heterologous expression of xylanase enzymes in lipogenic yeast Yarrowia lipolytica.

To develop a direct microbial sugar conversion platform for the production of lipids, drop-in fuels and chemicals from cellulosic biomass substrate, we chose Yarrowia lipolytica as a viable demonstration strain. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing sugars to produce lipids; however, it lacks the lignocellulose-degrading enzymes needed to break down biomass directly. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. The XynII and XlnD expressing Yarrowia strains exhibited an ability to grow on xylan mineral plates. This was shown by Congo Red staining of halo zones on xylan mineral plates. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action in converting xylan to xylose was observed when XlnD acted in concert with XynII. The successful expression of these xylanases in Yarrowia further advances us toward our goal to develop a direct microbial conversion process using this organism.

Abstract A06: Interspecific recombination between orthologous human and mouse BAC clones in E. coli: Exploring scalable humanization of cancer-relevant genes in the mouse genome

Abstract Our interests lie in humanizing specific regions of the mouse genome using large segments of human DNA with extents of 10s to 100s of kilobase pairs. To that end, we have undertaken a series of projects to transfer large segments of human DNA, from human BAC clones, into the orthologous region of corresponding mouse BAC clones using— (1), traditional recombineering methods; (2), recombinase-mediated cassette exchange (RCME); and (3), a novel technology that we call trans- or trimolecular-recombineering. In each instance the goal is to place human DNA within the larger context of homologous BAC-derived mouse arms in E. coli. Completed constructs will be used to drive homologous recombination in mouse embryonic stem (ES) cells with positive selection imparted at each end of the transferred human segment using dual, recombinase recognition site-flanked, selectable marker cassettes. Properly targeted ES cells will be used to develop viable mouse strains carrying the humanized segments. Selection cassettes will be removed by crossing to mouse strains with germline expression of the appropriate recombinases. All experiments have been designed to maintain a fixed genetic background from community-standard C57BL/6 strains. Our poster presentation details the specifics of our projects and describes progress to date. This research is supported by National Cancer Institute Grant Number 5P30CA034196-28. Citation Format: Tiffany Leidy-Davis, David E. Bergstrom. Interspecific recombination between orthologous human and mouse BAC clones in E. coli: Exploring scalable humanization of cancer-relevant genes in the mouse genome. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A06.

English

In order to acquire a high-producing rennet strain from Bacillus, different microwave irradiation intensities and durations were tested. The effect of microwave irradiation power and time were studied and the microwave mutagenesis parameters were optimized. The results indicated that the best conditions for microwave mutagenesis that produced better performed mutant strains were 450 W, 4 min. Under these conditions a viable strain with high-producing rennet (Strain No.: BsB1.1) was achieved whose genetic stability is good and rennet activity reaches 516.12 U/g which has increased by 29.0% compared with the original strain in the experiment. Key words: Bacillus subtilis, high-producing rennet, microwave mutagenesis.

Pathogenic potential of Saccharomyces strains isolated from dietary supplements.

Saccharomyces cerevisiae plays a beneficial role in health because of its intrinsic nutritional value and bio-functional properties, which is why it is also used as a dietary supplement. However, the perception that S. cerevisiae is harmless has changed due to an increasing number of infections caused by this yeast. Given this scenario, we have tested whether viable strains contained in dietary supplements displayed virulence-associated phenotypic traits that could contribute to virulence in humans. We have also performed an in vivo study of the pathogenic potential of these strains using a murine model of systemic infection by intravenous inoculation. A total of 5 strains were isolated from 22 commercial products and tested. Results highlight one strain (D14) in terms of burden levels in brains and kidneys and ability to cause death, whereas the other two strains (D2 and D4) were considered of low virulence. Our results suggest a strong relationship between some of the virulence-associated phenotypic traits (ability to grow at 39°C and pseudohyphal growth) and the in vivo virulence in a mouse model of intravenous inoculation for isolates under study. The isolate displaying greatest virulence (D14) was evaluated in an experimental murine model of gastrointestinal infection with immunosuppression and disruption of mucosal integrity, which are common risk factors for developing infection in humans, and results were compared with an avirulent strain (D23). We showed that D14 was able to spread to mesenteric nodes and distant organs under these conditions. Given the widespread consumption of dietary supplements, we recommend only safe strains be used.

Scientific Opinion on the safety and efficacy of fumonisin esterase (FUMzyme®) as a technological feed additive for pigs

FUMzyme® is an enzyme-based feed additive intended to degrade fumonisin mycotoxins found as contaminants in feeds for growing pigs. It is produced from a genetically modified strain of Komagataella pastoris. The viable production strain and its recombinant genes are not present in the final product. Piglets showed no adverse effect when given feed containing 15-times the maximum recommended dose. Consequently the FEEDAP Panel concludes that the additive is safe for pigs when used within the dose range proposed (15 – 300 units/kg feed). In studies with the additive, no evidence of mutagenicity or genotoxicity was detected and no evidence of toxicity found in a subchronic oral toxicity study. Metabolites of fumonisin B1 produced by the action of the active agent have a lower toxicity than the parent compound. Consequently use of the additive in feed for pigs is considered safe for consumers of pork products. The additive is non-irritant and is not a skin sensitiser but presents a hazard for respiratory sensitisation. The active substance of FUMzyme® is a protein and as such is largely degraded/inactivated during passage through the digestive tract of animals. In addition, any hydrolysis of fumonisins in the digestive tract would anticipate that which would occur naturally in soils. Therefore, no risks for the environment are expected. A number of short and long-term feeding studies were made in which pigs were given feed contaminated with fumonisins with or without the additive. Each included a measure of the ratio of sphinganine/sphingosine, considered the most sensitive endpoint for fumonisin toxicosis. This ratio was significantly reduced in piglets by the addition of FUMzyme® at the minimum proposed dose when added to diets contaminated with fuminosins above and below the guidance limits operating in the EU. Sphinganine/sphingosine values generally returned to those seen in animals fed corresponding diets free from fumonisins.

Effect of multispecies probiotic supplements on serum minerals, liver enzymes and blood pressure in patients with type 2 diabetes

Emerging evidence suggests that diabetes is associated with altering serum minerals, elevated liver enzymatic activity and blood pressures. This study was designed to determine the effects of multispecies probiotic supplements on serum minerals, liver enzymes and blood pressure in diabetic patients. This randomized double-blinded controlled clinical trial was performed among 58 diabetic patients aged 35–70 y. Subjects were randomly assigned to consume either multispecies probiotic supplements (N = 28) or the placebo group (N = 30) for 8 weeks. The multispecies probiotic supplement was consisted of seven viable and freeze-dried strains: Lactobacillus acidophilus (2 × 109 CFU), Lactobacillus casei (7 × 109 CFU), Lactobacillus rhamnosus (1.5 × 109 CFU), Lactobacillus bulgaricus (2 × 108 CFU), Bifidobacterium breve (2 × 1010 CFU), Bifidobacterium longum (7 × 109 CFU), Streptococcus thermophilus (1.5 × 109 CFU) and 100 mg fructo-oligosaccharide with lactose as carrier substances. Fasting blood samples were taken at baseline and after 8-week intervention to measure serum minerals, liver enzymes and total bilirubin. Consumption of the probiotic supplements, compared to the placebo, resulted in an increased serum calcium concentrations (0.21 vs. −0.83 mg/dL, P = 0.009) and a decreased serum alanine aminotransferase (ALT) levels (−2.46 vs. 4.62 mg/dL, P = 0.02). We did not find a significant difference in terms of effect on serum magnesium, zinc, iron, alkaline phosphatase (ALP), aspartate aminotransferase (AST) levels and blood pressures comparing the two groups. In conclusion, multispecies probiotic supplementation among diabetic patients had beneficial effects on serum calcium and ALT concentrations.

Detection of Ralstonia solanacearum from Asymptomatic Tomato Plants, Irrigation Water, and Soil Through Non-selective Enrichment Medium with hrp Gene-Based Bio-PCR

Bacterial wilt of tomato caused by Ralstonia solanacearum (Smith) Yabuuchi et al. (Microbiol Immunol 39:897-904, 1995) is a serious disease, which causes losses up to 60 % depending on environmental conditions, soil property, and cultivars. In present investigation, nucleotide sequences of virulence, hypersensitive response and pathogenicity (hrp) gene were used to design a pair of primer (Hrp_rs 2F: 5'-AGAGGTCGACGCGATACAGT-3' and Hrp_rs 2R: 5'-CATGAGCAAGGACGAAGTCA-3') for amplification of bacterial genome. The genomic DNA of 27 isolates of R. solanacearum race 1 biovar 3 & 4 was amplified at 323 bp. The specificity of primer was tested on 13 strains of R. solanacearum with other group of bacteria such as Xanthomonas oryzae pv. oryzae, X. campestris pv. campestris, and X. citri subsp. citri. Primer amplified DNA fragment of R. solanacearum at 323 bp. The sensitivity of the primer was 200 cfu/ml and improved further detection level by using non-specific enrichment medium casamino acids-pepton-glucose broth followed by PCR (BIO-PCR). Out of 130 samples of asymptomatic tomato plants, irrigation water, and soil collected from bacterial wilt infested field in different agro-climatic regions of India, R. solanacearum was detected from 86.9, 88.5, and 90.9 per cents samples using BIO-PCR, respectively. The primer was found specific for detecting viable and virulent strains of R. solanacearum and useful for the diagnosis of R. solanacearum in tomato seedlings and monitoring of pathogen in irrigation water and soil.

Sustainable Saffron Production as Influenced by Integrated Nitrogen Management in Typic Hapludalfs of NW Himalayas

The effects of integrated nitrogen management (INM) on saffron yield, corm production, nutrient concentration, crocin content, and soil health were studied in field experiments at the Dryland (Karewa) Agriculture Research Station, Budgam District of Kashmir Himalayas, India, during 2006–2010. The levels of fertilizers applied were 0, 45, and 90 kg ha−1 of nitrogen; 0, 30, and 60 t ha−1 of farm yard manure (FYM), and 0 and 5 kg ha −1 of Azotobacter in solid form. The greatest yields of 3.64 and 3.51 kg ha−1 were observed when nitrogen was applied at 90 kg ha−1 and FYM was applied at 60 t ha−1. The increases over the controls (2.31 and 2.45 kg ha−1) were 57.57% and 43.26%, respectively. The maximum corm productions (10.26 and 13.10 t ha−1) were observed with the application of nitrogen at 90 kg ha−1 and FYM at 60 t ha−1 respectively, with the corresponding increases of 79.62% and 260.97% over their respective controls. Biofertilizer application in the form of viable strain of Azotobacter significantly increased the corm production only. The influence of INM on nutrient and crocin content of saffron and soil health was also found to be sustainable over nonapplication of organic and inorganic fertilizers.

Occurrence of an Environmental Acinetobacter baumannii Strain Similar to a Clinical Isolate in Paleosol from Croatia

Over the past decade, bacteria of the genus Acinetobacter have emerged as a leading cause of hospital-acquired infections. Outbreaks of Acinetobacter infections are considered to be caused exclusively by contamination and transmission in hospital environments. The natural habitats of clinically important multiresistant Acinetobacter spp. remain to be defined. In this paper, we report an incidental finding of a viable multidrug-resistant strain of Acinetobacter baumannii, related to clinical isolates, in acid paleosol from Croatia. The environmental isolate of A. baumannii showed 87% similarity to a clinical isolate originating from a hospital in this geographic area and was resistant to gentamicin, trimethoprim-sulfamethoxazole, ciprofloxacin, and levofloxacin. In paleosol, the isolate was able to survive a low pH (3.37), desiccation, and a high temperature (50°C). The probable source of A. baumannii in paleosol is illegally disposed waste of external origin situated in the abandoned quarry near the sampling site. The bacteria could have been leached from waste by storm water and thus infiltrated the paleosol.

High-Resolution Profiling of Stationary-Phase Survival Reveals Yeast Longevity Factors and Their Genetic Interactions

Lifespan is influenced by a large number of conserved proteins and gene-regulatory pathways. Here, we introduce a strategy for systematically finding such longevity factors in Saccharomyces cerevisiae and scoring the genetic interactions (epistasis) among these factors. Specifically, we developed an automated competition-based assay for chronological lifespan, defined as stationary-phase survival of yeast populations, and used it to phenotype over 5,600 single- or double-gene knockouts at unprecedented quantitative resolution. We found that 14% of the viable yeast mutant strains were affected in their stationary-phase survival; the extent of true-positive chronological lifespan factors was estimated by accounting for the effects of culture aeration and adaptive regrowth. We show that lifespan extension by dietary restriction depends on the Swr1 histone-exchange complex and that a functional link between autophagy and the lipid-homeostasis factor Arv1 has an impact on cellular lifespan. Importantly, we describe the first genetic interaction network based on aging phenotypes, which successfully recapitulated the core-autophagy machinery and confirmed a role of the human tumor suppressor PTEN homologue in yeast lifespan and phosphatidylinositol phosphate metabolism. Our quantitative analysis of longevity factors and their genetic interactions provides insights into the gene-network interactions of aging cells.

Molecular detection and analysis of a novel metalloprotease gene of entomopathogenic Serratia marcescens strains in infected Galleria mellonella

Serratia marcescens strains isolated from entomopathogenic nematodes (Rhabditis sp.) were examined for their pathogenicity and establishment in wax moth (Galleria mellonella) larvae. All the Serratia strains were potently pathogenic to G. mellonella larvae, leading to death within 48 h. The strains were shown to possess a metalloprotease gene encoding for a novel serralysin-like protein. Rapid establishment of the bacteria in infected larvae was confirmed by specific polymerase chain reaction (PCR) detection of a DNA fragment encoding for this protein. Detection of the viable Serratia strains in infected larvae was validated using the SYBR Green reverse transcriptase real-time PCR assay targeting the metalloprotease gene. Nucleotide sequences of the metalloprotease gene obtained in our study showed 72 single nucleotide polymorphisms (SNP) and 3 insertions compared with the metalloprotease gene of S. marcescens E-15. The metalloprotease gene had 60 synonymous and 8 nonsynonymous substitutions relative to the closest GenBank entry, S. marcescens E-15. A comparison of the amino acid composition of the new serralysin-like protein with that of the serralysin protein of S. marcescens E-15 revealed differences at 11 positions and a new aspartic acid residue. Analysis of the effect of protein variation suggests that a new aspartic acid residue resulting from nonsynonymous nucleotide mutations in the protein structure could have the most significant effect on its biological function. The new metalloprotease gene and (or) its product could have applications in plant agricultural biotechnology.

Gingipains from Porphyromonas gingivalis play a significant role in induction and regulation of CXCL8 in THP-1 cells.

BackgroundPorphyromonas gingivalis is an important bacterial etiological agent involved in periodontitis. The bacterium expresses two kinds of cysteine proteases called gingipains: arginine gingipains (RgpA/B) and lysine gingipain (Kgp). This study evaluated the interaction between P. gingivalis and THP-1 cells, a widely used monocytic cell line, in vitro with a focus on CXCL8 at the gene and protein levels and its fate thereafter in cell culture supernatants. THP-1 cells were stimulated with viable and heat-killed wild-type strains ATCC 33277 or W50 or viable isogenic gingipain mutants of W50, E8 (Rgp mutant) or K1A (Kgp mutant), for 24 hours.ResultsELISA and qPCR results show an elevated CXCL8 expression and secretion in THP-1 cells in response to P. gingivalis, where the heat-killed ATCC33277 and W50 induced higher levels of CXCL8 in comparison to their viable counterparts. Furthermore, the Kgp-deficient mutant K1A caused a higher CXCL8 response compared to the Rgp-deficient E8. Chromogenic quantification of lipopolysaccharide (LPS) in supernatant showed no significant differences between viable and heat killed bacteria except that W50 shed highest levels of LPS. The wild-type strains secreted relatively more Rgp during the co-culture with THP-1 cells. The CXCL8 degradation assay of filter-sterilized supernatant from heat-killed W50 treated cells showed that Rgp was most efficient at CXCL8 hydrolysis. Of all tested P. gingivalis strains, adhesion and internalization in THP-1 cells was least conspicuous by Rgp-deficient P. gingivalis (E8), as demonstrated by confocal imaging.ConclusionsW50 and its Kgp mutant K1A exhibit a higher immunogenic and proteolytic function in comparison to the Rgp mutant E8. Since K1A differs from E8 in the expression of Rgp, it is rational to conclude that Rgp contributes to immunomodulation in a more dynamic manner in comparison to Kgp. Also, W50 is a more virulent strain when compared to the laboratory strain ATCC33277.

Establishment and application of a flow cytometry-based rapid drug susceptibility test for Candida species

Objective To establish a flow cytometry-based drug susceptibility test for the rapid de-tection of antifungal susceptibility or resistance of Candida isolates.Methods The gate selection and opti-mal experimental conditions of flow cytometry-based drug susceptibility test were determined by using Candi-da albicans strain ATCC90029 as the test strain and propidium iodide （ PI） as the fluorescent dye .The es-tablished flow cytometry-based drug susceptibility test was used to detect the susceptibility or resistance to fluconazole or voriconazole of 110 isolates belonging to Candida species, and the obtained results were com-pared with those by using typical M 27-A3 constant dilution method .Results The killed and viable Candida albicans ATCC90029 strains were clearly divided into two groups on the figure of SS /log （FL3） by regulating voltages.There was a high correlation between the results of susceptibility test and the proportions of killed and viable fungi in mixture （r=0.999）.The flow cytometry-based drug susceptibility test could provide the results within 30 min and its optimal concentration of fungal suspension , time of drug-fungus incubation , dyeing method and time were 1.0×106/ml, 3 h incubation and sodium deoxycholate-pretreated plus PI dye-ing for 5 min, respectively .The total coincident rates between the established test and the constant dilution method were 98.2%and 87.3%in the detection of drug susceptibility of 110 fungal isolates to fluconazole and voriconazole .Conclusion The flow cytometry-based drug susceptibility test shows advantages of rapidi-ty, accuracy and high sensitivity compared with the constant dilution method .It has a great potential for clin-ical application . Key words: Flow cytometry; Drug susceptibility test ; Candida; Fluconazole ; Voriconazole

Accelerated discovery via a whole-cell model

Whole-cell modeling promises to facilitate scientific inquiry by prioritizing future experiments based on existing datasets. To test this promise, we compared simulated growth rates with new measurements for all viable single-gene disruption strains in Mycoplasma genitalium. The discrepancies between simulations and experiments led to novel model predictions about specific kinetic parameters that we subsequently validated. These findings represent the first application of whole-cell modeling to accelerate biological discovery.