Contribution of trehalose to the salinity and high temperature tolerance of <i>C. salexigens</i>.

<p>(A) Effect of salinity on the growth of <i>C. salexigens</i> ectoines and/or trehalose defective mutants. Cells of <i>C. salexigens</i> CHR61 (▪), Δ<i>ectABC</i>::Tn<i>1732</i> (•), <i>otsA</i>::Ω (□) and Δ<i>ectABC</i>::Tn<i>1732 otsA</i>::Ω (○) strains were grown at 37°C in M63 minimal medium with 0.75 M NaCl. (B) Effect of temperature on the growth of <i>C. salexigens</i> hydroxyectoine and/or trehalose defective mutants. Cells of <i>C. salexigens</i> CHR61 (▪), <i>ectD</i>::Ω (▴), <i>otsA</i>::Ω (□), and <i>ectD</i>::Ω <i>otsA</i>::Ω (▵) strains were grown at 45°C in M63 minimal medium with 2.5 M NaCl. Values shown are the mean of two replicas of each condition in three independent experiments ± SD (standard deviation).</p

Survival of <i>E. coli</i> and <i>C. salexigens</i> strains after vacuum-drying and subsequent storage at 28°C.

<p>The values are means ± standard errors.</p>1<p>Desiccation under vacuum.</p>2<p>Desiccation under vacuum with temperature ramping.</p

Regulation of trehalose synthesis by <i>C. salexigens</i> in response to temperature and salinity stress.

<p>(A) Accumulation of trehalose by <i>C. salexigens</i> CHR61 in response to temperature and salinity. Cells were grown in minimal medium M63 at 37°C (with 0.75 and 2.5 M NaCl) and 45°C (with 2.5 M NaCl), up to early stationary phase. Trehalose content was measured colorimetrically as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033587#s2" target="_blank">Materials and Methods</a>. For each determination, a growth curve under the same condition used to measure trehalose accumulation is shown. Histograms representing trehalose accumulation are shown above the sampling time. The trehalose values are the mean of three replicas of each condition in two independent experiments ± SD (standard deviation). <i>C. salexigens</i> does not grow at 45°C with 0.75 M NaCl. (B) relative <i>otsA</i> mRNA levels determined by quantitative PCR (qPCR) in <i>C. salexigens</i> CHR61 strain. Total RNA was extracted from cells grown in minimal medium M63 under the same conditions of temperature and salinity as above, and <i>otsA</i> mRNA abundance was measured by quantitative PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033587#s2" target="_blank">Materials and Methods</a>. The data are expressed in relative units and were estimated by the 2^−ΔCt method using the 16S rRNA gene as an internal control to normalize expression in each sample. <u>Results are shown as the fold change in expression relative to that of 0.75 M 37°C.</u> Real-time PCR quantification was performed twice, using RNA samples from independent cultures, and the values are the means standard deviations of three replicates from two independent experiments.</p

OtsA is involved in trehalose synthesis by <i>C. salexigens</i>.

<p>Genetic <i>con</i>text of the <i>C. salexigens treha</i>lose synthesis genes <i>otsA</i> (encoding a trehalose-6-phosphate synthase) and <i>otsB</i> (encoding a trehalose-6-phosphate phosphatase). Numbers into brackets denote intergenic regions. The arrow upstream of <i>csal0239</i> denotes a predicted α⁷⁰-dependent promoter. For the generation of <i>otsA</i>::Ω mutant strains, <i>otsA</i> was inactivated by the insertion of an Ω cassette, which carries resistance genes for streptomycin/spectinomycin, into its unique site <i>Sma</i>I (Sm), giving the plasmid pMO3.</p

Natural abundance ¹³C-NMR spectrum of major cytosolic solutes of the <i>C. salexigens</i> wild-type and <i>otsA</i> strains.

<p>CHR61control (A) and <i>otsA</i>:: (CHR185) (B) strains were grown in M63 minimal medium at 45°C with 2.5 M NaCl. Cells were extracted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033587#s2" target="_blank">Materials and Methods</a>. The major solutes were hydroxyectoine (H), ectoine (E), trehalose (T), glutamate (G) and Nγ-acetyldiaminobutyric acid (N).</p

New 4‑Acyl-1-phenylaminocarbonyl-2-phenylpiperazine Derivatives as Potential Inhibitors of Adenovirus Infection. Synthesis, Biological Evaluation, and Structure–activity Relationships

The search for human adenovirus (HAdV)-specific antiviral drugs for the treatment of HAdV infections in immunocompromised patients continues to be a challenging goal for medicinal chemistry. Here, we report the synthesis, biological evaluation, and structure–activity relationships of a small molecules library. We have identified six phenylpiperazine derivatives that significantly inhibited HAdV infection. These six compounds showed the capacity to block HAdV and, in addition, human cytomegalovirus (HCMV) replications at low micromolar concentration, with little or no cytotoxicity. On the basis of our biological studies, these molecules block HAdV and HCMV infections in different phases of their life cycle, providing potential candidates for the development of a new family of antiviral drugs for the treatment of infections by DNA viruses

THDP17 Decreases Ammonia Production through Glutaminase Inhibition. A New Drug for Hepatic Encephalopathy Therapy

<div><p>Ammonia production is implicated in the pathogenesis of hepatic encephalopathy (HE), being intestinal glutaminase activity the main source for ammonia. Management of ammonia formation can be effective in HE treatment by lowering intestinal ammonia production. The use of glutaminase inhibitors represents one way to achieve this goal. In this work, we have performed a search for specific inhibitors that could decrease glutaminase activity by screening two different groups of compounds: i) a group integrated by a diverse, highly pure small molecule compounds derived from thiourea ranging from 200 to 800 Daltons; and ii) a group integrated by commonly use compounds in the treatment of HE. Results shown that THDP-17 (10 µM), a thiourea derivate product, could inhibit the intestinal glutaminase activity (57.4±6.7%). Inhibitory effect was tissue dependent, ranging from 40±5.5% to 80±7.8% in an uncompetitive manner, showing V_max and K_m values of 384.62 µmol min⁻¹, 13.62 mM with THDP-17 10 µM, respectively. This compound also decreased the glutaminase activity in Caco-2 cell cultures, showing a reduction of ammonia and glutamate production, compared to control cultures. Therefore, the THDP-17 compound could be a good candidate for HE management, by lowering ammonia production.</p></div

Metformin inhibits glutaminase activity in a dose dependent manner: from 17.5% at 10 mM up to 68% at 100 mM (A) (* p<0.05).

<p>This effect, analyzed by Dixon plot, showed competitive inhibition kinetics, with a K_i of 14.28 mM (B). In Caco2 cells, metformin 20 mM showed 24% inhibition of glutaminase activity at 72 hours compared to control cultures (p<0.05) (glutamate production was decreased from 26.85±0.74 µM to 19.9±2.05 µM; p<0.05) (C).</p

<i>In vivo</i> inhibitory effect on glutaminase activity at different concentrations of THDP17 and DON (6-Diazo-5-oxo-L-norleucine) was tested in Caco-2 cell cultures.

<p>PAG inhibition by THDP-17 in cell cultures is effective at concentration higher than 5 µM, showing an inhibition of 18±2.1% and 46±3.4% at 20 and 100 µM, respectively (* p<0.05).</p

THDP-17 was administered orally after 12 h fasted mice.

<p>Animals were divided in 4 groups and deaths after treatment were recorded.</p><p>THDP-17 was administered orally after 12 h fasted mice.</p