Silencing of an efflux pump coding gene decreases the efflux rate of pyrazinoic acid in Mycobacterium smegmatis

Background: Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB). The recommended treatment for TB is based on the use of first-line drugs, including pyrazinamide (PZA). PZA is also a drug used in the treatment of multidrug-resistant TB (MDR-TB) because of its main effect against the latent stage. The main cause of resistance to PZA is mutations in the pncA gene, which compromise the activity of the encoded enzyme pyrazinamidase (PZAse), which hydrolyzes PZA into POA, the active antituberculosis molecule. The mechanism of action of PZA requires that POA is expelled from the bacterium by an efflux mechanism. After that, if the extracellular medium is sufficiently acidic, POA is protonated and returns to the cytosol, releasing the proton and repeating the cycle, resulting lethal to the bacteria. The efflux pump responsible for extruding the POA to the extracellular environment is not yet known. Mycobacterium smegmatis is naturally resistant to PZA and has a 900-fold faster POA efflux rate than MTB, and has the advantage to be a faster growing mycobacterium. Methods: In the present study we have silenced the transcription of several genes encoding efflux pumps in M. smegmatis by CRISPRi (CRISPR interference). These genes (MSMEG_0250, MSMEG_3815, MSMEG_0241, MSMEG_5046 and MSMEG_0410) were homologous to efflux pump genes in MTB. POA efflux rate was measured, and a quantitative Wayne's test was performed after silencing each gene. Results: Silencing of MSMEG_0250, resulted in an approximately 5-fold decrease in the POA efflux rate in M. smegmatis (P<0.0001). None of the other silenced genes showed a notable decrease in the POA efflux rate.

Ultrasound-targeted simvastatin-loaded microbubble destruction promotes OA cartilage repair by modulating the cholesterol efflux pathway mediated by PPARγ in rabbits

Aims To evaluate the effect of ultrasound-targeted simvastatin-loaded microbubble destruction (UTMD SV ) for alleviation of the progression of osteoarthritis (OA) in rabbits through modulation of the peroxisome proliferator-activated receptor (PPARγ). Methods In vitro, OA chondrocytes were treated with ultrasound (US), US-targeted microbubble destruction (UTMD), simvastatin (SV), and UTMD SV on alternate days for four weeks. Chondrocytes were also treated with PPARγ inhibitor, PPARγ inhibitor+ UTMD SV , and UTMD SV . The cholesterol efflux rate and triglyceride levels were measured using an assay kit and oil red O staining, respectively. In vivo, the OA rabbits were treated with a single intra-articular injection of UTMD, SV, and UTMD SV every seven days for four weeks. Cartilage histopathology was assessed by safranin-O staining and the Mankin score. Total cholesterol (TC) and high-density lipoprotein-cholesterol (HDL-C) in rabbit knee synovial fluid were detected by enzyme-marker assay. Aggrecan, collagen II, and PPARγ expression levels were analyzed by Western blotting (WB). Results In vitro, UTMD SV significantly increased the cholesterol efflux rate and aggrecan, collagen II, and PPARγ levels in OA chondrocytes; these effects were blocked by the PPARγ inhibitor. In vivo, UTMD SV significantly increased aggrecan, collagen II, PPARγ, and HDL-C levels, while TC levels and Mankin scores were decreased compared with the UTMD, SV, OA, and control groups. Conclusion UTMD SV promotes cartilage extracellular matrix synthesis by modulating the PPARγ-mediated cholesterol efflux pathway in OA rabbits. Cite this article: Bone Joint Res 2021;10(10):693–703.

Ultrasound-targeted Simvastatin-loaded Microbubble Destruction Promotes OA Cartilage Repair by Modulating Cholesterol Efflux Pathway Mediated by PPARγ in Rabbits.

Objective: To evaluate ultrasound-targeted simvastatin-loaded microbubble destruction (UTMDSV) attenuation of osteoarthritis (OA) progression in rabbits through modulation of the peroxisome proliferator-activated receptor (PPARγ)-mediated cholesterol efflux pathway.Methods: In vitro, chondrocytes were treated with ultrasound (US), US-targeted microbubble destruction (UTMD), simvastatin (SV) and UTMDsv on alternate days for 4 weeks. Chondrocytes were also treated with PPARγ inhibitor, PPARγ inhibitor+UTMDsv and UTMDsv. The cholesterol efflux rate and triglyceride were measured respectively by assay kit and oil red O staining. In vivo, the OA rabbits were treated with a single intra-articular injection of UTMD, SV and UTMDSV every 7 days for 4 weeks. Cartilage histopathology was assessed by safranin-O staining and the Mankin score. Total cholesterol (TC) and high-density lipoprotein-cholesterol (HDL-C) in rabbit knee synovial fluid were detected by enzyme-marker assay. Aggrecan, collagen II and PPARγ expression levels were analyzed western blotting (WB).Results: OA models exhibited primarily by a loss of aggrecan and collagen II, changes to subchondral bone architecture and cartilage degradation. In vitro, UTMDSV significantly increased the cholesterol efflux rate and aggrecan, collagen II and PPARγ levels in OA chondrocytes; these effects were blocked by the PPARγ inhibitor. In vivo, UTMDSV significantly increased aggrecan, collagen II, PPARγ and HDL-C levels, while TC levels and Mankin scores were decreased compared with the UTMD, SV, OA and control groups (95% CI: 0.069 to 6.671).Conclusion: UTMDSV promotes the expression of aggrecan and collagen II and relieve cartilage degradation by modulating the PPARγ-mediated cholesterol efflux pathway in rabbits.

The Influence and Mechanism of Paeoniflorin and Albiflorin on Strychnine and Brucine Transport Through Madin-Darby Canine Kidney/Multidrug Resistance1 Cells In Vitro Model

This research sought to study the influence and potentialmechanism of paeoniflorin and albiflorin on strychnine and brucine transport in MDCK-MDR1 cells regulated by P-gp. Cytotoxicity of drugs was tested by MTT assay, and the transport studies were performed in both directions in MDCK-MDR1 cells. The influence of drugs on P-gp ATPase, and the efflux function of P-gp, the expression levels of P-gp and MDR1 mRNA were also estimated. Strychnine and brucine showed well absorption, and the main transport mechanism might be passive diffusion. Verapamil could significantly decrease the efflux rate (ER) of strychnine and brucine, while the ER of strychnine and brucine was increased significantly when co-administrated with paeoniflorin or albiflorin, indicating that paeoniflorin and albiflorin could promote the efflux of these two alkaloids. Strychnine and brucine could activate the activity of P-gp ATPase, suppress the efflux function of P-gp, but have no significant effect on the expression of P-gp. In addition, strychnine could upregulate the expression of MDR1 mRNA. Paeoniflorin and albiflorin could increase the transmembrane transport of strychnine and brucine mediated by P-gp when co-administrated with strychnine or brucine via stimulating the activity of P-gp ATPase, enhancing the efflux function of P-g, increasing the expression levels of MDR1 mRNA and P-gp.

Uptake of Sulfate from Ambient Water by Freshwater Animals

To better understand how the sulfate (SO42−) anion may contribute to the adverse effects associated with elevated ionic strength or salinity in freshwaters, we measured the uptake and efflux of SO42− in four freshwater species: the fathead minnow (Pimephales promelas, Teleostei: Cyprinidae), paper pondshell (Utterbackia imbecillis, Bivalvia: Unionidae), red swamp crayfish (Procambarus clarkii, Crustacea: Cambaridae), and two-lined mayfly (Hexagenia bilineata, Insecta: Ephemeridae). Using δ(34S/32S) stable isotope ratios and the concentrations of S and SO42−, we measured the SO42− influx rate (Jin), net flux (Jnet), and efflux rate (Jout) during a 24 h exposure period. For all four species, the means of Jin for SO42− were positive, and Jin was significantly greater than 0 at both target SO42− concentrations in the fish and mollusk and at the lower SO42− concentration in the crayfish. The means of Jout and Jnet were much more variable than those for Jin, but several species by target SO42− concentration combinations for Jout and Jnet, were negative, which suggests the net excretion of SO42− by the animals. The results of our experiments suggest a greater regulation of SO42− in freshwater animals than has been previously reported.

Biochar combined with manure application can decrease organic matter decomposition compared to manure alone in the dry tropical cropland of south India

&lt;p&gt;Soils in the dry tropical croplands of south India are inherently low in soil carbon (C) stock, and it is essential to accumulate the soil C for sustainable soil management. Biochar is generally considered to be a useful material that enhance the soil C stock, though its real effect on soil C dynamics is still unclear especially in the dry tropical croplands such as south India. Thus, our objective was to evaluate the effect of biochar application on soil C dynamics for optimal soil management in south India. Field experiment was conducted in Tamil Nadu state (Inceptisols) from Sep. 2017 to Apr. 2019 (1.5 years), which include two times sorghum cultivation (each 4 months) with six treatment plots (control (C), biochar (B) (8.2 Mg C ha&lt;sup&gt;-1&lt;/sup&gt;), farmyard manure (FYM) (F) (1.1 Mg C ha&lt;sup&gt;-1&lt;/sup&gt;), chemical fertilizer (CF) (100 kg N; 40 kg P ha&lt;sup&gt;-1&lt;/sup&gt;), biochar and FYM (B+F), and biochar and chemical fertilizer (B+CF)). We applied biochar once at the beginning of the experiment to evaluate the effective duration of biochar in soil after application, while we applied FYM every year before crop cultivation. We periodically measured the CO&lt;sub&gt;2&lt;/sub&gt; efflux rate (29 times totally) with continuous environmental data including soil moisture (0-15 cm) and soil temperature (5 cm), and estimated the total CO&lt;sub&gt;2&lt;/sub&gt; flux as C output, based on the relationship between the CO&lt;sub&gt;2&lt;/sub&gt; efflux rate and environmental data. We found that the CO&lt;sub&gt;2&lt;/sub&gt; efflux rate in the B+F plot tended to be lower than the F plot throughout the experimental period, though the significant difference between the B+F plot and F plot was only in the cultivation period of the 1&lt;sup&gt;st&lt;/sup&gt; year, in case of using the analysis of variance for each cultivation period separately. We found that cumulative CO&lt;sub&gt;2&lt;/sub&gt; flux in the B+F plot (2.2 Mg C ha&lt;sup&gt;-1&lt;/sup&gt; 1.5 year&lt;sup&gt;-1&lt;/sup&gt;) was also lower than the F plot (2.5 Mg C ha&lt;sup&gt;-1&lt;/sup&gt; 1.5 year&lt;sup&gt;-1&lt;/sup&gt;), and that biochar and FYM application decreased ca. 0.3 Mg C ha&lt;sup&gt;-1&lt;/sup&gt; 1.5 year&lt;sup&gt;-1 &lt;/sup&gt;decomposition compared to the application of FYM alone. This might be because combined application of biochar and FYM decreased the soil microbial activity, resulting in the lower FYM decomposition in the B+F plot. Our results indicate that biochar combined with FYM application would effective for soil C sequestration, and hence for sustainable soil management in the dry tropical cropland.&lt;/p&gt;

The proton electrochemical gradient induces a kinetic asymmetry in the symport cycle of LacY

LacY catalyzes accumulation of galactosides against a concentration gradient by coupling galactoside and H+ transport (i.e., symport). While alternating access of sugar- and H+-binding sites to either side of the membrane is driven by binding and dissociation of sugar, the electrochemical H+ gradient (∆μ∼H+) functions kinetically by decreasing the Km for influx 50- to 100-fold with no change in Kd. The affinity of protonated LacY for sugar has an apparent pK (pKapp) of ∼10.5, due specifically to the pKa of Glu325, a residue that plays an irreplaceable role in coupling. In this study, rates of lactose/H+ efflux were measured from pH 5.0 to 9.0 in the absence or presence of a membrane potential (ΔΨ, interior positive), and the effect of the imposed ΔΨ on the kinetics of efflux was also studied in right-side-out membrane vesicles. The findings reveal that ∆μ∼H+ induces an asymmetry in the transport cycle based on the following observations: 1) the efflux rate of WT LacY exhibits a pKapp of ∼7.2 that is unaffected by the imposed ΔΨ; 2) ΔΨ increases the rate of efflux at all tested pH values, but enhancement is almost 2 orders of magnitude less than observed for influx; 3) mutant Glu325 ˗ Ala does little or no efflux in the absence or presence of ΔΨ, and ambient pH has no effect; and 4) the effect of ΔΨ (interior positive) on the Km for efflux is almost insignificant relative to the 50- to 100-fold decrease in the Km for influx driven by ΔΨ (interior negative).

A Glycine max sodium/hydrogen exchanger enhances salt tolerance through maintaining higher Na+ efflux rate and K+/Na+ ratio in Arabidopsis

Background Soybean (Glycine max (L.)) is one the most important oil-yielding cash crops. However, the soybean production has been seriously restricted by salinization. It is therefore crucial to identify salt tolerance-related genes and reveal molecular mechanisms underlying salt tolerance in soybean crops. A better understanding of how plants resist salt stress provides insights in improving existing soybean varieties as well as cultivating novel salt tolerant varieties. In this study, the biological function of GmNHX1, a NHX-like gene, and the molecular basis underlying GmNHX1-mediated salt stress resistance have been revealed. Results We found that the transcription level of GmNHX1 was up-regulated under salt stress condition in soybean, reaching its peak at 24 h after salt treatment. By employing the virus-induced gene silencing technique (VIGS), we also found that soybean plants became more susceptible to salt stress after silencing GmNHX1 than wild-type and more silenced plants wilted than wild-type under salt treatment. Furthermore, Arabidopsis thaliana expressing GmNHX1 grew taller and generated more rosette leaves under salt stress condition compared to wild-type. Exogenous expression of GmNHX1 resulted in an increase of Na+ transportation to leaves along with a reduction of Na+ absorption in roots, and the consequent maintenance of a high K+/Na+ ratio under salt stress condition. GmNHX1-GFP-transformed onion bulb endothelium cells showed fluorescent pattern in which GFP fluorescence signals enriched in vacuolar membranes. Using the non-invasive micro-test technique (NMT), we found that the Na+ efflux rate of both wild-type and transformed plants after salt treatment were significantly higher than that of before salt treatment. Additionally, the Na+ efflux rate of transformed plants after salt treatment were significantly higher than that of wild-type. Meanwhile, the transcription levels of three osmotic stress-related genes, SKOR, SOS1 and AKT1 were all up-regulated in GmNHX1-expressing plants under salt stress condition. Conclusion Vacuolar membrane-localized GmNHX1 enhances plant salt tolerance through maintaining a high K+/Na+ ratio along with inducing the expression of SKOR, SOS1 and AKT1. Our findings provide molecular insights on the roles of GmNHX1 and similar sodium/hydrogen exchangers in regulating salt tolerance.

Metallochaperones Are Needed for Mycobacterium tuberculosis and Escherichia coli Nicotinamidase-Pyrazinamidase Activity

ABSTRACT Mycobacterium tuberculosis nicotinamidase-pyrazinamidase (PZAse) is a metalloenzyme that catalyzes conversion of nicotinamide-pyrazinamide to nicotinic acid-pyrazinoic acid. This study investigated whether a metallochaperone is required for optimal PZAse activity. M. tuberculosis and Escherichia coli PZAses (PZAse-MT and PZAse-EC, respectively) were inactivated by metal depletion (giving PZAse-MT–Apo and PZAse-EC–Apo). Reactivation with the E. coli metallochaperone ZnuA or Rv2059 (the M. tuberculosis analog) was measured. This was repeated following proteolytic and thermal treatment of ZnuA and Rv2059. The CDC1551 M. tuberculosis reference strain had the Rv2059 coding gene knocked out, and PZA susceptibility and the pyrazinoic acid (POA) efflux rate were measured. ZnuA (200 μM) achieved 65% PZAse-EC–Apo reactivation. Rv2059 (1 μM) and ZnuA (1 μM) achieved 69% and 34.3% PZAse-MT–Apo reactivation, respectively. Proteolytic treatment of ZnuA and Rv2059 and application of three (but not one) thermal shocks to ZnuA significantly reduced the capacity to reactivate PZAse-MT–Apo. An M. tuberculosis Rv2059 knockout strain was Wayne positive and susceptible to PZA and did not have a significantly different POA efflux rate than the reference strain, although a trend toward a lower efflux rate was observed after knockout. The metallochaperone Rv2059 restored the activity of metal-depleted PZAse in vitro. Although Rv2059 is important in vitro, it seems to have a smaller effect on PZA susceptibility in vivo. It may be important to mechanisms of action and resistance to pyrazinamide in M. tuberculosis. Further studies are needed for confirmation. IMPORTANCE Tuberculosis is an infectious disease caused by the bacterium Mycobacterium tuberculosis and remains one of the major causes of disease and death worldwide. Pyrazinamide is a key drug used in the treatment of tuberculosis, yet its mechanism of action is not fully understood, and testing strains of M. tuberculosis for pyrazinamide resistance is not easy with the tools that are presently available. The significance of the present research is that a metallochaperone-like protein may be crucial to pyrazinamide’s mechanisms of action and of resistance. This may support the development of improved tools to detect pyrazinamide resistance, which would have significant implications for the clinical management of patients with tuberculosis: drug regimens that are appropriately tailored to the resistance profile of a patient’s individual strain lead to better clinical outcomes, reduced onward transmission of infection, and reduction of the development of resistant strains that are more challenging and expensive to treat.