In Vitro Activity of Mitochondrial ATP Synthetase Inhibitors Against Plasmodium falciparum

ABSTRACT. The mitochondrion appears to be essential for the growth of asexual, intraerythrocytic stages of Plasmodium falciparum and may thus be a suitable chemotherapeutic target. The in vitro activity of almitrine, a mitochondrial ATP synthetase inhibitor used for the treatment of hypoxemia, was compared with other mitochondrial inhibitors against chloroquine-susceptible and chloroquine-resistant P. falciparum using an isotopic semimicro drug susceptibility assay. The 50% inhibitory concentration (IC50) values of almitrine (range: 2.6–19.8 μM) were within similar range of values of other mitochondrial ATP synthetase inhibitors and doxycycline, a mitochondrial protein synthesis inhibitor. Almitrine was equally active against chloroquine-susceptible and chloroquine-resistant parasites. Drug combination studies showed no interaction between chloroquine and almitrine. Our results suggest that almitrine, a clinically safe drug, may represent a lead compound with a specific target against the mitochondrial ATP synthetase which may be useful for antimalarial chemotherapy.     

The Influence of Efflux Pump Inhibitors on the Activity of Non-Antibiotic NSAIDS against Gram-Negative Rods

Background

Most patients with bacterial infections suffer from fever and various pains that require complex treatments with antibiotics, antipyretics, and analgaesics. The most common drugs used to relieve these symptoms are non-steroidal anti-inflammatory drugs (NSAIDs), which are not typically considered antibiotics. Here, we investigate the effects of NSAIDs on bacterial susceptibility to antibiotics and the modulation of bacterial efflux pumps.

Methodology

The activity of 12 NSAID active substances, paracetamol (acetaminophen), and eight relevant medicinal products was analyzed with or without pump inhibitors against 89 strains of Gram-negative rods by determining the MICs. Furthermore, the effects of NSAIDs on the susceptibility of clinical strains to antimicrobial agents with or without PAβN (Phe-Arg-β-naphtylamide) were measured.

Results

The MICs of diclofenac, mefenamic acid, ibuprofen, and naproxen, in the presence of PAβN, were significantly (≥4-fold) reduced, decreasing to 25–1600 mg/L, against the majority of the studied strains. In the case of acetylsalicylic acid only for 5 and 7 out of 12 strains of P. mirabilis and E. coli, respectively, a 4-fold increase in susceptibility in the presence of PAβN was observed. The presence of Aspirin resulted in a 4-fold increase in the MIC of ofloxacin against only two strains of E. coli among 48 tested clinical strains, which included species such as E. coli, K. pneumoniae, P. aeruginosa, and S. maltophilia. Besides, the medicinal products containing the following NSAIDs, diclofenac, mefenamic acid, ibuprofen, and naproxen, did not cause the decrease of clinical strains’ susceptibility to antibiotics.

Conclusions

The effects of PAβN on the susceptibility of bacteria to NSAIDs indicate that some NSAIDs are substrates for efflux pumps in Gram-negative rods. Morever, Aspirin probably induced efflux-mediated resistance to fluoroquinolones in a few E. coli strains.     

Proton Pump Inhibitors and the Risk of Adverse Cardiac Events

Background

Recent evidence suggests that proton pump inhibitors (PPIs) might be linked with adverse cardiac events, but a causal relationship is unproven.

Methods

We applied the self-matched case series method to two studies using population-based health care data from Ontario, Canada between 1996 and 2008. The first included subjects aged 66 years or older hospitalized for acute myocardial infarction within 12 weeks following initiation of PPI, while the second included subjects hospitalized for heart failure. In both studies we designated the primary risk interval as the initial 4 weeks of therapy and the control interval as the final 4 weeks. To test the specificity of our findings we examined use of histamine H2 receptor antagonists and benzodiazepines, drugs with no plausible causal link to adverse cardiac events.

Results

During the 13-year study period, we identified 5550 hospital admissions for acute myocardial infarction and 6003 admissions for heart failure within 12 weeks of commencing PPI therapy. In the main analyses, we found that initiation of a PPI was associated with a higher risk of acute myocardial infarction (odds ratio 1.8; 95% confidence interval 1.7 to 1.9) and heart failure (odds ratio 1.8; 95% confidence interval 1.7 to 1.9). However, secondary analyses revealed similar risk estimates histamine H2 receptor antagonists and benzodiazepines, drugs with no known or suspected association with adverse cardiac events.

Conclusion

PPIs are associated with a short-term risk of adverse cardiac events, but similar associations are seen with other drugs exhibiting no known cardiac toxicity. Collectively these observations suggest that the association between PPIs and adverse cardiac events does not represent reflect cause-and-effect.     

Effects of Proton Pump Inhibitors on Lung Cancer Precise Radiotherapy-Induced Radiation Pneumonitis

The objective of this study was to explore the effects of proton pump inhibitors (PPIs) on the development and prognosis of lung cancer precise radiotherapy-induced radiation pneumonitis. Clinical materials of 84 lung cancer patients who had radiation pneumonitis after precise radiotherapy were retrospectively analyzed, and the patients were divided into PPI group and control group, according to whether or not PPIs were applied. The development and prognosis of patients and the effects of different doses of PPI on patient condition from two groups were compared. There were 57 PPI cases in PPI group and 27 cases in control group. Basic characteristics of patients were not statistically different between the two groups; however, white blood cell count, oxygenation indexes, blood gas pH, and lung imaging index were significantly different (p < 0.05), indicating that radiation pneumonitis tended to be more severe in PPI group. As regards effects of PPI on prognosis of two groups, remission rate of radiation pneumonia in PPI group was significantly less than that of the control group. Among 57 cases in PPI group, there were 31 patients applied with PPI ≤ 1DDD and 31 patients applied with PPI > 1DDD. In comparison of the various parameters of patients, 7 days after being applied with different doses of PPI, there were no significant differences between the parameters of radiation pneumonitis. PPIs should be cautiously utilized to avoid the effects of lung cancer radiotherapy-induced radiation pneumonia.     

In Vitro Activity of Doxycycline Against Bacteria from Clinical Material

Doxycycline (alpha-6-deoxy-oxytetracycline) was tested against various bacteria of recent clinical origin with 30-mug discs. The antibiotic susceptibility of these bacteria to commonly used antimicrobial agents was also established. Those bacteria which responded with equivocal zones of inhibition about the tetracycline compounds were tested by the tube dilution technique. Staphylococci and enterococci consistently displayed greater in vitro susceptibility to doxycycline than to tetracycline or demethylchlortetracycline.     

The Effect of Glyphosate on Potential Pathogens and Beneficial Members of Poultry Microbiota In Vitro

The use of glyphosate modifies the environment which stresses the living microorganisms. The aim of the present study was to determine the real impact of glyphosate on potential pathogens and beneficial members of poultry microbiota in vitro. The presented results evidence that the highly pathogenic bacteria as Salmonella Entritidis, Salmonella Gallinarum, Salmonella Typhimurium, Clostridium perfringens and Clostridium botulinum are highly resistant to glyphosate. However, most of beneficial bacteria as Enterococcus faecalis, Enterococcus faecium, Bacillus badius, Bifidobacterium adolescentis and Lactobacillus spp. were found to be moderate to highly susceptible. Also Campylobacter spp. were found to be susceptible to glyphosate. A reduction of beneficial bacteria in the gastrointestinal tract microbiota by ingestion of glyphosate could disturb the normal gut bacterial community. Also, the toxicity of glyphosate to the most prevalent Enterococcus spp. could be a significant predisposing factor that is associated with the increase in C. botulinum-mediated diseases by suppressing the antagonistic effect of these bacteria on clostridia.     

Inhibitory Activity of Honeysuckle Extracts against Influenza A Virus In Vitro and In Vivo

Virologica Sinica - Honeysuckle has been used in the treatment of influenza virus infection for thousands of years in China. However, its main active components and the functional mechanisms remain...     

Improving Antibiotic Activity against Wound Pathogens with Manuka Honey In Vitro

Following the discovery of synergistic action between oxacillin and manuka honey against methicillin-resistant Staphylococcus aureus, this study was undertaken to search for further synergistic combinations of antibiotics and honey that might have potential in treating wounds. Fifteen antibiotics were tested with and without sublethal concentrations of manuka honey against each of MRSA and Pseudomonas aeruginosa using disc diffusion, broth dilution, E strip, chequerboard titration and growth curves. Five novel antibiotic and manuka honey combinations were found that improved antibacterial effectiveness in vitro and these offer a new avenue of future topical treatments for wound infections caused by these two important pathogens.     

Comparative Antiviral Efficacy of Viral Protease Inhibitors against the Novel SARS-CoV-2 In Vitro

Virologica Sinica - The recent outbreak of novel coronavirus pneumonia (COVID-19) caused by a new coronavirus has posed a great threat to public health. Identifying safe and effective antivirals is...     

Non-peptidic Cruzain Inhibitors with Trypanocidal Activity Discovered by Virtual Screening and In Vitro Assay

A multi-step cascade strategy using integrated ligand- and target-based virtual screening methods was developed to select a small number of compounds from the ZINC database to be evaluated for trypanocidal activity. Winnowing the database to 23 selected compounds, 12 non-covalent binding cruzain inhibitors with affinity values (K _i) in the low micromolar range (3–60 µM) acting through a competitive inhibition mechanism were identified. This mechanism has been confirmed by determining the binding mode of the cruzain inhibitor Nequimed176 through X-ray crystallographic studies. Cruzain, a validated therapeutic target for new chemotherapy for Chagas disease, also shares high similarity with the mammalian homolog cathepsin L. Because increased activity of cathepsin L is related to invasive properties and has been linked to metastatic cancer cells, cruzain inhibitors from the same library were assayed against it. Affinity values were in a similar range (4–80 µM), yielding poor selectivity towards cruzain but raising the possibility of investigating such inhibitors for their effect on cell proliferation. In order to select the most promising enzyme inhibitors retaining trypanocidal activity for structure-activity relationship (SAR) studies, the most potent cruzain inhibitors were assayed against T. cruzi-infected cells. Two compounds were found to have trypanocidal activity. Using compound Nequimed42 as precursor, an SAR was established in which the 2-acetamidothiophene-3-carboxamide group was identified as essential for enzyme and parasite inhibition activities. The IC₅₀ value for compound Nequimed42 acting against the trypomastigote form of the Tulahuen lacZ strain was found to be 10.6±0.1 µM, tenfold lower than that obtained for benznidazole, which was taken as positive control. In addition, by employing the strategy of molecular simplification, a smaller compound derived from Nequimed42 with a ligand efficiency (LE) of 0.33 kcal mol⁻¹ atom⁻¹ (compound Nequimed176) is highlighted as a novel non-peptidic, non-covalent cruzain inhibitor as a trypanocidal agent candidate for optimization.     

Effects of Proton Pump Inhibitors on the Gastrointestinal Microbiota in Gastroesophageal Reflux Disease

Proton pump inhibitors(PPIs) are commonly used to lessen symptoms in patients with gastroesophageal reflux disease(GERD). However, the effects of PPI therapy on the gastrointestinal microbiota in GERD patients remain unclear. We examined the association between the PPI usage and the microbiota present in gastric mucosal and fecal samples from GERD patients and healthy controls(HCs) using 16 S rRNA gene sequencing. GERD patients taking PPIs were further divided into short-term and long-term PPI user groups. We showed that PPI administration lowered the relative bacterial diversity of the gastric microbiota in GERD patients. Compared to the non-PPIuser and HC groups, higher abundances of Planococcaceae, Oxalobacteraceae, and Sphingomonadaceae were found in the gastric microbiota from the PPI-user group. In addition, the Methylophilus genus was more highly abundant in the long-term PPI user group than in the short-term PPI-user group. Despite the absence of differences in alpha diversity, there were significant differences in the fecal bacterial composition of between GERD patients taking PPIs and those not taking PPIs. There was a higher abundance of Streptococcaceae, Veillonellaceae, Acidaminococcaceae,Micrococcaceae, and Flavobacteriaceae present in the fecal microbiota from the PPI-user group than those from the non-PPI-user and HC groups. Additionally, a significantly higher abundance of Ruminococcus was found in GERD patients on long-term PPI medication than that on shortterm PPI medication. Our study indicates that PPI administration in patients with GERD has a significant effect on the abundance and structure of the gastric mucosal microbiota but only on the composition of the fecal microbiota.     

In vitro Antifungal Activity of Essential oils and Major Components against Fungi Plant Pathogens

The indiscriminate use of synthetic fungicides has caused several problems to the environment, which place human and animal health at risk. Due to this fact, the search for natural alternatives to control phytopathogenic fungi growth has increased. This study aims to evaluate the antifungal activity of two essential oils (EOs) and three major components of EOs on the radial growth and spore germination of Fusarium verticillioides and Alternaria tenuissima. Minimum and half‐maximal inhibitory concentrations (CMI and CI₅₀) at 96 h for each treatment were calculated. Lemongrass EO and citral caused the highest inhibition for A. tenuissima (CMI of 1000 μl/l and CI₅₀ of 10 μl/l). For F. verticillioides, the most effective component was geraniol (CMI and CI₅₀ of 1000 and 250 μl/l, respectively). Spore germination rate was delayed by the EOs and major components tested. The use of EOs was effective to control these two fungal species in their different grow stages.     

In Vivo and In Vitro Activity by Diverse Chelators against Trypanosoma brucei brucei1

A system of prescreens and screen has been developed to select chelators as potential drugs against Trypanosoma brucei brucei EATRO 110. The chelators tested were nearly all commercially available, low molecular, and having moderate to high affinity for Fe(III). We prescreened 70 compounds showing heme-sparing or inhibitory activity in a Crithidia fasciculata growth system having excess Fe and minimal hemin. Of these, 45 were highly trypanocidal for suspensions of bloodstream T. b. brucei; criteria of activity here were immobilization, lysis, and loss of infectivity. Eighteen of the chelators highly active in the suspension prescreen were tried in T. b. brucei-infected mice. Thirteen of these chelators were curative in mice with 24-h infections, that is, they allowed survival >30 days beyond the untreated controls. 3,4-Dihydroxycinnamic acid (caffeic acid). 2,9-dimethyl-1, 10 phenanthroline (neocuproine), and 2-pyridinecarboxaldehyde-2-pyridyl-hydrazone cured five out of five mice after an i.v. dose of 100 mg/kg. Salicylaldehyde thiosemicarbazone cured five out of five mice at an i.p. dose of 500 mg/kg. Lesser activity was shown by several other chelators.     

Effect of Proton Pump Inhibitors on Serum Thyroid-Stimulating Hormone Level in Euthyroid Patients Treated with Levothyroxine for Hypothyroidism

ObjectiveTo examine retrospectively the effect of proton pump inhibitors (PPIs) on thyrotropin (thyroid-stimulating hormone or TSH) values in patients with hypothyroidism and normal TSH levels receiving levothyroxine (LT₄) replacement therapy.MethodsThe data collection was done by retrospective review of electronic medical records from the period of December 2002 to August 2005 from patients with hypothyroidism who were receiving at least 25 μg of LT₄ replacement daily at Queens Hospital Center. The first 92 patients meeting all inclusion and exclusion criteria were included in the study. The study group (N = 37) patient data were collected by selecting euthyroid patients who had received stable LT₄ replacement for at least 6 months and in whom PPI therapy (lansoprazole) was later initiated. TSH levels were collected before and at least 2 months after the PPI treatment was started. The control group (N = 55) patient data were collected by reviewing TSH levels among euthyroid patients with a history of hypothyroidism receiving stable LT₄ therapy and not receiving a PPI during the period of data collection. The statistical analysis was done by comparing the mean change in TSH level in each group with use of the Student t test.ResultsIn the study group, the mean change in the TSH level from before to at least 2 months after initiation of PPI therapy, 0.69 ± 1.9 μIU/mL, was statistically significant (P = 0.035). In the control group, the mean change in the TSH level during the study period, 0.11 ± 1.06 μIU/mL, was not statistically significant (P = 0.45).ConclusionTo our best knowledge, this is the first study in humans with hypothyroidism demonstrating the effect of PPIs on serum TSH levels. PPIs should be added to the list of medications affecting the level of thyroid hormone in patients with hypothyroidism treated with LT₄ replacement. Patients with hypothyroidism and normal TSH values during LT₄ replacement therapy may need additional thyroid function testing after treatment with PPIs and may need adjustment of their LT₄ dose. (Endocr Pract. 2007;13:345-349)     

Proton Fluxes and the Activity of a Stelar Proton Pump in Onion Roots

The xylem vessels of excised adventitious roots of onion, Alliumcepa, were perfused with unbuffered nutrient solution adjustedinitially to either pH 9·3 or 3·9; the pH of thesolution after passage through the xylem, at rates not lessthan 2 xylem volume changes min^–1, was close to pH 6·5in both instances. The flux of H⁺ across the xylem/symplastboundary into mildly alkaline, phosphate-buffered solutionsperfusing the vessels could be increased greatly with increasingbuffer strength, up to a maximum value between 0·5–1·0pmol H⁺ mm^–2 s^–1. The apparent neutralization ofacidic malic acid buffers had a slightly lower maximum capacity,equivalent to –0·3 to –0·5 pmol H⁺mm^–2 s^–1. The addition of 5·0 pmol m^–3fusicoccin (FC) to the xylem perfusion solution stimulated theentry of H⁺ into the xylem; in unbuffered perfusion solutionsthe pH fell to pH 3·6 after a lag of 25–35 min.FC additions to phosphate-buffered solutions also stimulatedthe H⁺ flux to an extent similar to that in unbuffered solution,viz. 0·2–0·4 pmol mm^–2 s^–1. The release of K⁺ (³⁶Rb-labelled) into xylem sap transientlyincreased as the [K⁺] in weakly buffered perfusion solutionswas raised stepwise; a very marked increase being seen whenthe concentration was raised to 100 mol m^–3 from 40 molm^–3. The addition of 5·0 mmol m^–3 FC to theperfusing solution containing 100 mol m^–3 K⁺ rapidly decreasedthe K⁺ flux to the xylem as the H⁺ flux increased. Fusicoccinalso inhibited the flux of K⁺ into unbuffered perfusion solutionsbut the effect appeared reversible. Addition of 10 mmol m^–3abscisic acid (ABA) to the perfusion solution quickly producedtransient increases in both K⁺ and H⁺ fluxes into the xylem.In this and other experiments using weakly phosphate-bufferedperfusing solutions, H⁺ fluxes were comparable in size to thoseof K⁺ The results are consistent with the idea that the stele of onionroots contains a proton trarislocating ATPase whose activityresponds to the pH of the xylem sap. It is evident that theactivity of the proton secreting and proton neutralizing mechanismsin the xylem parenchyma control the movement of other ions acrossthe xylem/symplast boundary. Key words: Xylem perfusion, fusicoccin, abscisic acid, pH gradient     

Antiviral Activity of Trappin-2 and Elafin In Vitro and In Vivo against Genital Herpes

Serine protease inhibitor elafin (E) and its precursor, trappin-2 (Tr), have been associated with mucosal resistance to HIV-1 infection. We recently showed that Tr/E are among principal anti-HIV-1 molecules in cervicovaginal lavage (CVL) fluid, that E is ∼130 times more potent than Tr against HIV-1, and that Tr/E inhibited HIV-1 attachment and transcytosis across human genital epithelial cells (ECs). Since herpes simplex virus 2 (HSV-2) is a major sexually transmitted infection and risk factor for HIV-1 infection and transmission, we assessed Tr/E contribution to defense against HSV-2. Our in vitro studies demonstrated that pretreatment of endometrial (HEC-1A) and endocervical (End1/E6E7) ECs with human Tr-expressing adenovirus (Ad/Tr) or recombinant Tr/E proteins before or after HSV-2 infection resulted in significantly reduced virus titers compared to those of controls. Interestingly, E was ∼7 times more potent against HSV-2 infection than Tr. Conversely, knockdown of endogenous Tr/E by small interfering RNA (siRNA) significantly increased HSV-2 replication in genital ECs. Recombinant Tr and E reduced viral attachment to genital ECs by acting indirectly on cells. Further, lower viral replication was associated with reduced secretion of proinflammatory interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α) and decreased NF-κB nuclear translocation. Additionally, protected Ad/Tr-treated ECs demonstrated enhanced interferon regulatory factor 3 (IRF3) nuclear translocation and increased antiviral IFN-β in response to HSV-2. Lastly, in vivo studies of intravaginal HSV-2 infection in Tr-transgenic mice (Etg) showed that despite similar virus replication in the genital tract, Etg mice had reduced viral load and TNF-α in the central nervous system compared to controls. Collectively, this is the first experimental evidence highlighting anti-HSV-2 activity of Tr/E in female genital mucosa.