Effect of active vitamin D on proliferation, cell cycle and apoptosis in endometriotic stromal cells.

RESEARCH QUESTION: What is the effect of vitamin D3 (1,25(OH)2D3) on proliferation, cell cycle and apoptosis of endometrial stromal cells (ESC) in endometriotic patients? DESIGN: ESC isolated from 10 women with endometriosis and 10 healthy controls were treated with 1,25(OH)2D3. The proliferation of control endometrial stromal cells (CESC), eutopic endometrial stromal cells (EuESC) and ectopic endometrial stromal cells (EESC) was analysed 72 h after the treatment using methyl thiazolyl tetrazolium assay. Propidium iodide staining and flow cytometry were used to determine the cell cycle distribution in ESC. Annexin V/propidium iodide double staining was used to evaluate apoptosis in ESC. RESULTS: In the presence of oestrogen, 1,25(OH)2D3 treatment inhibited the proliferation of ESC from all three origins (P = 0.009 for CESC, P = 0.005 for EuESC and P < 0.001 for EESC). The percentage of S phase cells in EESC was higher than in EuESC and CESC (P = 0.002 and P = 0.001, respectively). The percentage of S phase cells in EuESC was higher than in CESC (P = 0.005). The percentage of G1 phase cells in EESC was lower than that of EuESC and CESC (P = 0.003 and P = 0.002, respectively) and the percentage of G1 phase cells in EuESC was lower than that of CESC (P = 0.007). Moreover, 1,25(OH)2D3 inhibited cell cycle regardless of cell type (P = 0.002 in EESC, P = 0.001 in EuESC and P = 0.014 in CESC), but in the absence of oestrogen, inhibited cell cycle only in EuESC (P = 0.012). CONCLUSIONS: Although 1,25(OH)2D3 increased apoptotic and necrotic cells and decreased live cells in the EuESC and EESC, it did not affect apoptosis in CESC and only increased necrotic cells. These findings indicate that 1,25(OH)2D3 potentially has a growth-inhibiting and pro-apoptotic effect on ESC from endometriotic patients.

Effect of cholesterol-loaded cyclodextrin treatment of bovine sperm on capacitation timing.

Pre-loading bovine sperm with cholesterol prior to freezing is known to increase cryosurvival, though the timing of capacitation in these sperm has not been evaluated. The objective of this study was to determine if there is a potential delay in capacitation timing in these sperm due to the increased cholesterol content. Flow cytometric evaluation was utilized to assess viability, and stain technology to assess acrosome intactness (Propidium Iodide/FITC-PNA), intracellular calcium levels (Propidium Iodide/FLUO 3-AM) and membrane fluidity (Merocyanine 540/YO-PRO-1). Cholesterol-loaded cyclodextrin (CLC) (2 mg/mL) improved post-thaw viability to 61% from 45% in control sperm (p < .05). The addition of ionomycin (0.05 mM) induced capacitation in sperm by 1 h, resulting in increased intracellular calcium and increased acrosome reaction, and consequently viability loss by 3 h. Treatment with CLC significantly decreased membrane fluidity in sperm (p < .05). In conclusion, CLC-treated sperm required 1 h more to capacitate when compared with non-treated sperm based on percentage of live cells with high membrane disorder (p < .05). Increased cryosurvival and viability over time was observed, but longer time to capacitate may hinder fertilization capacity and/or require adjustments to timing of in vitro fertilization.

Lipopeptide surfactin ameliorates the cell uptake of platensimycin and enhances its therapeutic effect on treatment of MRSA skin infection.

OBJECTIVES: The rapid development of drug-resistant bacteria, especially MRSA, poses severe threats to global public health. Adoption of antibiotic adjuvants has proved to be one of the efficient ways to solve such a crisis. Platensimycin and surfactin were comprehensively studied to combat prevalent MRSA skin infection. METHODS: MICs of platensimycin, surfactin or their combinations were determined by resazurin assay, while the corresponding MBCs were determined by chequerboard assay. Growth inhibition curves and biofilm inhibition were determined by OD measurements. Membrane permeability analysis was conducted by propidium iodide staining, and morphological characterizations were performed by scanning electron microscopy. Finally, the therapeutic effects on MRSA skin infections were evaluated in scald-model mice. RESULTS: The in vitro assays indicated that surfactin could significantly improve the antibacterial performance of platensimycin against MRSA, especially the bactericidal activity. Subsequent mechanistic studies revealed that surfactin not only interfered with the biofilm formation of MRSA, but also disturbed their cell membranes to enhance membrane permeability, and therefore synergistically ameliorated MRSA cellular uptake of platensimycin. Further in vivo assessment validated the synergistic effect of surfactin on platensimycin and the resultant enhancement of therapeutical efficacy in MRSA skin-infected mice. CONCLUSIONS: The combination of effective and biosafe surfactin and platensimycin could be a promising and efficient treatment for MRSA skin infection, which could provide a feasible solution to combat the major global health threats caused by MRSA.

Disruption of mitochondrial functions involving mitochondrial permeability transition pore opening caused by maleic acid in rat kidney.

Propionic acid (PA) predominantly accumulates in tissues and biological fluids of patients affected by propionic acidemia that may manifest chronic renal failure along development. High urinary excretion of maleic acid (MA) has also been described. Considering that the underlying mechanisms of renal dysfunction in this disorder are poorly known, the present work investigated the effects of PA and MA (1-5 mM) on mitochondrial functions and cellular viability in rat kidney and cultured human embryonic kidney (HEK-293) cells. Mitochondrial membrane potential (∆ψm), NAD(P)H content, swelling and ATP production were measured in rat kidney mitochondrial preparations supported by glutamate or glutamate plus malate, in the presence or absence of Ca2+. MTT reduction and propidium iodide (PI) incorporation were also determined in intact renal cells pre-incubated with MA or PA for 24 h. MA decreased Δψm and NAD(P)H content and induced swelling in Ca2+-loaded mitochondria either respiring with glutamate or glutamate plus malate. Noteworthy, these alterations were fully prevented by cyclosporin A plus ADP, suggesting the involvement of mitochondrial permeability transition (mPT). MA also markedly inhibited ATP synthesis in kidney mitochondria using the same substrates, implying a strong bioenergetics impairment. In contrast, PA only caused milder changes in these parameters. Finally, MA decreased MTT reduction and increased PI incorporation in intact HEK-293 cells, indicating a possible association between mitochondrial dysfunction and cell death in an intact cell system. It is therefore presumed that the MA-induced disruption of mitochondrial functions involving mPT pore opening may be involved in the chronic renal failure occurring in propionic acidemia.

In Vitro Study of Cytotoxic Mechanisms of Alkylphospholipids and Alkyltriazoles in Acute Lymphoblastic Leukemia Models.

This study investigates the efficacy of miltefosine, alkylphospholipid, and alkyltriazolederivative compounds against leukemia lineages. The cytotoxic effects and cellular and molecular mechanisms of the compounds were investigated. The inhibitory potential and mechanism of inhibition of cathepsins B and L, molecular docking simulation, molecular dynamics and binding free energy evaluation were performed to determine the interaction of cathepsins and compounds. Among the 21 compounds tested, C9 and C21 mainly showed cytotoxic effects in Jurkat and CCRF-CEM cells, two human acute lymphoblastic leukemia (ALL) lineages. Activation of induced cell death by C9 and C21 with apoptotic and necrosis-like characteristics was observed, including an increase in annexin-V+propidium iodide-, annexin-V+propidium iodide+, cleaved caspase 3 and PARP, cytochrome c release, and nuclear alterations. Bax inhibitor, Z-VAD-FMK, pepstatin, and necrostatin partially reduced cell death, suggesting that involvement of the caspase-dependent and -independent mechanisms is related to cell type. Compounds C9 and C21 inhibited cathepsin L by a noncompetitive mechanism, and cathepsin B by a competitive and noncompetitive mechanism, respectively. Complexes cathepsin-C9 and cathepsin-C21 exhibited significant hydrophobic interactions, water bridges, and hydrogen bonds. In conclusion, alkyltriazoles present cytotoxic activity against acute lymphoblastic lineages and represent a promising scaffold for the development of molecules for this application.

6,6'-((Methylazanedyl)bis(methylene))bis(2,4-dimethylphenol) Induces Autophagic Associated Cell Death through mTOR-Mediated Autophagy in Lung Cancer.

Autophagy is the multistep mechanism for the elimination of damaged organelles and misfolded proteins. This mechanism is preceded and may induce other program cell deaths such as apoptosis. This study unraveled the potential pharmacological effect of 24MD in inducing the autophagy of lung cancer cells. Results showed that 24MD was concomitant with autophagy induction, indicating by autophagosome staining and the induction of ATG5, ATG7 and ubiquitinated protein, p62 expression after 12-h treatment. LC3-I was strongly conversed to LC3-II, and p62 was downregulated after 24-h treatment. The apoptosis-inducing activity was found after 48-h treatment as indicated by annexin V-FITC/propidium iodide staining and the activation of caspase-3. From a mechanistic perspective, 24-h treatment of 24MD at 60 µM substantially downregulated p-mTOR. Meanwhile, p-PI3K and p-Akt were also suppressed by 24MD at concentrations of 80 and 100 µM, respectively. We further confirmed m-TOR-mediated autophagic activity by comparing the effect of 24MD with rapamycin, a potent standard mTOR1 inhibitor through Western blot and immunofluorescence assays. Although 24MD could not suppress p-mTOR as much as rapamycin, the combination of rapamycin and 24MD could increase the mTOR suppressive activity and LC3 activation. Changing the substituent groups (R groups) from dimethylphenol to ethylphenol in EMD or changing methylazanedyl to cyclohexylazanedyl in 24CD could only induce apoptosis activity but not autophagic inducing activity. We identified 24MD as a novel compound targeting autophagic cell death by affecting mTOR-mediated autophagy.

JNC-1043, a Novel Podophyllotoxin Derivative, Exerts Anticancer Drug and Radiosensitizer Effects in Colorectal Cancer Cells.

The objective of this study was to determine whether (5S)-5-(4-benzyloxy-3,5-dimethoxy-phenyl)-5,9-dihydro-8H-furo [3',4':6,7] naphtho [2,3-d] [1,3]dioxol-6-one (JNC-1043), which is a novel chemical derivative of ß-apopicropodophyllin, acts as a novel potential anticancer reagent and radiosensitizer in colorectal cancer (CRC) cells. Firstly, we used MTT assays to assess whether JNC-1043 could inhibit the cell proliferation of HCT116 and DLD-1 cells. The IC50 values of these cell lines were calculated as 114.5 and 157 nM, respectively, at 72 h of treatment. Using doses approximating the IC50 values, we tested whether JNC-1043 had a radiosensitizing effect in the CRC cell lines. Clonogenic assays revealed that the dose-enhancement ratios (DER) of HCT116 and DLD-1 cells were 1.53 and 1.25, respectively. Cell-counting assays showed that the combination of JNC-1043 and γ-ionizing radiation (IR) enhanced cell death. Treatment with JNC-1043 or IR alone induced cell death by 50~60%, whereas the combination of JNC-1043 and IR increased this cell death by more than 20~30%. Annexin V-propidium iodide assays showed that the combination of JNC-1043 and IR increased apoptosis by more 30~40% compared to that induced by JNC-1043 or IR alone. DCFDA- and MitoSOX-based assays revealed that mitochondrial ROS production was enhanced by the combination of JNC-1043 and IR. Finally, we found that suppression of ROS by N-acetylcysteine (NAC) blocked the apoptotic cell death induced by the combination of JNC-1043 and IR. The xenograft model also indicated that the combination of JNC-1043 and IR increased apoptotic cell death in tumor mass. These results collectively suggest that JNC-1043 acts as a radiosensitizer and exerts anticancer effects against CRC cells by promoting apoptosis mediated by mitochondrial ROS.

Chitooligosaccharide prevents vascular endothelial cell apoptosis by attenuation of endoplasmic reticulum stress via suppression of oxidative stress through Nrf2-SOD1 up-regulation.

CONTEXT: Endoplasmic reticulum (ER) stress contributes to endothelium pathological conditions. Chitooligosaccharides (COS) have health benefits, but their effect on endothelial cells is unknown. We demonstrate for the first time a protective effect of COS against ER-induced endothelial cell damage. OBJECTIVE: To evaluate the protective effect of COS on ER stress-induced apoptosis in endothelial cells. MATERIAL AND METHODS: Endothelial (EA.hy926) cells were pre-treated with COS (250 or 500 µg/mL) for 24 h, and then treated with 0.16 µg/mL of Tg for 24 h and compared to the untreated control. Apoptosis and necrosis were detected by Annexin V-FITC/propidium iodide co-staining. Reactive oxygen species (ROS) were measured with the DCFH2-DA and DHE probes. The protective pathway and ER stress markers were evaluated by reverse transcription-polymerase chain reaction, western blot, and immunofluorescence analyses. RESULTS: COS attenuated ER stress-induced cell death. The viability of EA.hy926 cells treated with Tg alone was 44.97 ± 1% but the COS pre-treatment increased cells viability to 74.74 ± 3.95% in the 250 µg/mL COS and 75.34 ± 2.4% in the 500 µg/mL COS treatments. Tg induced ER stress and ROS, which were associated with ER stress-mediated death. Interestingly, COS reduced ROS by upregulating nuclear factor-E2-related factor 2 (Nrf2), and the oxidative enzymes, superoxide dismutase1 (SOD1) and catalase. COS also suppressed up-regulation of the ER-related apoptosis protein, CHOP induced by Tg. CONCLUSIONS: COS protected against ER stress-induced apoptosis in endothelial cells by suppressing ROS and up-regulation Nrf2 and SOD1. These findings support the use of COS to protect endothelial cells.

Trimethylamine oxide induces pyroptosis of vascular endothelial cells through ALDH2/ROS/NLRP3/GSDMD pathway. / 氧化三甲胺通过ALDH2/ROS/NLRP3/GSDMDé€šè·¯è¯±å¯¼è¡€ç®¡å† çš®ç»†èƒžç„¦äº¡ï¼ˆè‹±æ–‡ï¼‰.

OBJECTIVES: Trimethylamine oxide (TMAO) is a metabolite of intestinal flora and is known to promote the progression of atherosclerotic plaques. However, how TMAO works, including its effect on vascular endothelial cells, is not fully understood. This study aims to explore the biological role of TMAO in human umbilical vein endothelial cells (HUVECs) and the underlying mechanism. METHODS: Cell pyroptosis and the loss of plasma membrane integrity were induced under TMAO stimulation in HUVECs. The plasma membrane integrity of the cells was measured by Hoechst 33342/propidium iodide (PI) staining and lactate dehydrogenase leakage assay, and the changes in cell morphology were observed by atomic force microscope. The expression of proteins related to pyroptosis was determined by Western blotting or immunofluorescence. Mitochondrial acetaldehyde dehydrogenase 2 (ALDH2) activity in HUVECs was measured by the ALDH2 activity assay kit, and the level of reactive oxygen species (ROS) was detected by fluorescent probe DCFH-DA. RESULTS: TMAO induced pyroptotic cell death, manifesting by the presence of propidium iodide-positive cells, the leakage of lactate dehydrogenase, the production of N-terminal gasdermin D (GSDMD-N), and the formation of plasma membrane pores. Moreover, TMAO induced elevated expression of inflammasome components, nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1 in cells. TMAO significantly inhibited ALDH2 activity and increased intracellular ROS production. However, the activation of ALDH2 by pharmacological manipulation attenuated TMAO-induced inflammasome activation and GSDMD-N production. CONCLUSIONS: TMAO induces pyroptosis of vascular endothelial cells through the ALDH2/ROS/NLRP3/GSDMD signaling pathway, which may be a potential therapeutic target for improving the treatment of atherosclerosis.

[Effects of arsenic and its main metabolites on A549 cell apoptosis and the expression of pro-apoptotic genes Bad and Bik].

Objective: To investigate the effect of arsenic and its main metabolites on the apoptosis of human lung adenocarcinoma cell line A549 and the expression of pro-apoptotic genes Bad and Bik. Methods: In October 2020, A549 cells were recovered and cultured, and the cell viability was detected by the cell counting reagent CCK-8 to determine the concentration and time of sodium arsenite exposure to A549. The study was divided into NaAsO(2) exposure groups and metobol: le expoure groups: the metabolite comparison groups were subdivided into the control group, the monomethylarsinic acid exposure group (60 µmol/L) , and the dimethylarsinic acid exposure group (60 µmol/L) ; sodium arsenite dose groups were subdivided into 4 groups: control group (0) , 20, 40, 60 µmol/L sodium arsenite NaAsO(2). Hoechst 33342/propidium iodide double staining (Ho/PI) was used to observe cell apoptosis and real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of Bad and Bik mRNA in cells after exposure. Western blotting was used to detect the protein expressions of Bad, P-Bad-S112, Bik, cleaved Bik and downstream proteins poly ADP-ribose polymerase PARP1 and cytochrome C (Cyt-C) , using spectrophotometry to detect the activity changes of caspase 3, 6, 8, 9. Results: Compared with the control group, the proportion of apoptotic cells in the 20, 40, and 60 µmol/L NaAsO(2) dose groups increased significantly (P<0.01) , and the expression levels of Bad, Bik mRNA, the protein expression levels of Bad, P-Bad-S112, Bik, cleaved Bik, PARP1, Cyt-C were increased (all P<0.05) , and the activities of Caspase 3, 6, 8, and 9 were significantly increased with significantly differences (P<0.05) . Compared with the control group, the expression level of Bad mRNA in the DMA exposure group (1.439±0.173) was increased with a significant difference (P=0.024) , but there was no significant difference in the expression level of Bik mRNA (P=0.788) . There was no significant differences in the expression levels of Bad and Bik mRNA in the poison groups (P=0.085, 0.063) . Compared with the control group, the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to MMA were 0.696±0.023, 0.707±0.014, 0.907±0.031, 1.032±0.016, and there was no significant difference between the two groups (P=0.469, 0.669, 0.859, 0.771) ; the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to DMA were 0.698±0.030, 0.705±0.022, 0.908±0.015, 1.029±0.010, and there was no difference between the two groups (P=0.479, 0.636, 0.803, 0.984) . Conclusion: Sodium arsenite induces the overexpression of Bad and Bik proteins, initiates the negative feedback regulation of phosphorylated Bad and the degradation of Bik, activates the downstream proteins PARP1, Cyt-C and Caspase pathways, and mediates the apoptosis of A549 cells.

Human polymorphisms in GSDMD alter the inflammatory response.

Exomic studies have demonstrated that innate immune genes exhibit an even higher degree of variation than the majority of other gene families. However, the phenotypic implications of this genetic variation are not well understood, with effects ranging from hypomorphic to silent to hyperfunctioning. In this work, we study the functional consequences of this variation by investigating polymorphisms in gasdermin D, the key pyroptotic effector protein. We find that, although SNPs affecting potential posttranslational modifications did not affect gasdermin D function or pyroptosis, polymorphisms disrupting sites predicted to be structurally important dramatically alter gasdermin D function. The manner in which these polymorphisms alter function varies from conserving normal pyroptotic function to inhibiting caspase cleavage to disrupting oligomerization and pore formation. Further, downstream of inflammasome activation, polymorphisms that cause loss of gasdermin D function convert inflammatory pyroptotic cell death into immunologically silent apoptotic cell death. These findings suggest that human genetic variation can alter mechanisms of cell death in inflammation.

Ethidium and propidium monoazide: comparison of potential toxicity on Vibrio sp. viability.

Vibrio sp., ubiquitous in the aquatic ecosystem, are bacteria of interest because of their involvement in human health, causing gastroenteritis after ingestion of seafood, as well as their role in vibriosis leading to severe losses in aquaculture production. Their ability to enter a viable but non-culturable (VBNC) state under stressful environmental conditions may lead to underestimation of the Vibrio population by traditional microbiological enumeration methods. As a result, using molecular methods in combination with EMA or PMA allows the detection of viable (VBNC and culturable viable) cells. In this study, the impact of the EMA and PMA was tested at different concentrations on the viability of several Vibrio species. We compared the toxicity of these two DNA-binding dyes to determine the best pretreatment to use with qPCR to discriminate between viable and dead Vibrio cells. Our results showed that EMA displayed lethal effects for each strain of V. cholerae and V. vulnificus tested. In contrast, the concentrations of PMA tested had no toxic effect on the viability of Vibrio cells studied. These results may help to achieve optimal PMA-qPCR methods to detect viable Vibrio sp. cells in food and environmental samples.

KD025 Shifts Pulmonary Endothelial Cell Bioenergetics and Decreases Baseline Lung Permeability.

KD025 is a ROCK2 inhibitor currently being tested in clinical trials for the treatment of fibrotic lung diseases. The therapeutic effects of KD025 are partly due to its inhibition of profibrotic pathways and fat metabolism. However, whether KD025 affects pulmonary microvascular endothelial cell (PMVEC) function is unknown, despite evidence that alveolar-capillary membrane disruption constitutes major causes of death in fibrotic lung diseases. We hypothesized that KD025 regulates PMVEC metabolism, pH, migration, and survival, a series of interrelated functional characteristics that determine pulmonary barrier integrity. We used PMVECs isolated from Sprague Dawley rats. KD025 dose-dependently decreased lactate production and glucose consumption. The inhibitory effect of KD025 was more potent compared with other metabolic modifiers, including 2-deoxy-glucose, extracellular acidosis, dichloroacetate, and remogliflozin. Interestingly, KD025 increased oxidative phosphorylation, whereas 2-deoxy-glucose did not. KD025 also decreased intracellular pH and induced a compensatory increase in anion exchanger 2. KD025 inhibited PMVEC migration, but fasudil (nonspecific ROCK inhibitor) did not. We tested endothelial permeability in vivo using Evans Blue dye in the bleomycin pulmonary fibrosis model. Baseline permeability was decreased in KD025-treated animals independent of bleomycin treatment. Under hypoxia, KD025 increased PMVEC necrosis as indicated by increased lactate dehydrogenase release and propidium iodide uptake and decreased ATP; it did not affect Annexin V binding. ROCK2 knockdown had no effect on PMVEC metabolism, pH, and migration, but it increased nonapoptotic caspase-3 activity. Together, we report that KD025 promotes oxidative phosphorylation; decreases glycolysis, intracellular pH, and migration; and strengthens pulmonary barrier integrity in a ROCK2-independent manner.

High-throughput screening yields several small-molecule inhibitors of repeat-associated non-AUG translation.

Repeat-associated non-AUG (RAN) translation is a noncanonical translation initiation event that occurs at nucleotide-repeat expansion mutations that are associated with several neurodegenerative diseases, including fragile X-associated tremor ataxia syndrome (FXTAS), ALS, and frontotemporal dementia (FTD). Translation of expanded repeats produces toxic proteins that accumulate in human brains and contribute to disease pathogenesis. Consequently, RAN translation constitutes a potentially important therapeutic target for managing multiple neurodegenerative disorders. Here, we adapted a previously developed RAN translation assay to a high-throughput format to screen 3,253 bioactive compounds for inhibition of RAN translation of expanded CGG repeats associated with FXTAS. We identified five diverse small molecules that dose-dependently inhibited CGG RAN translation, while relatively sparing canonical translation. All five compounds also inhibited RAN translation of expanded GGGGCC repeats associated with ALS and FTD. Using CD and native gel analyses, we found evidence that three of these compounds, BIX01294, CP-31398, and propidium iodide, bind directly to the repeat RNAs. These findings provide proof-of-principle supporting the development of selective small-molecule RAN translation inhibitors that act across multiple disease-causing repeats.

A long-amplicon quantitative PCR assay with propidium monoazide to enumerate viable Listeria monocytogenes after heat and desiccation treatments.

The objective of this study was to develop a qPCR method for specific enumeration of viable Listeria monocytogenes in food processing facilities and heat treated products. Primers specific for L. monocytogenes were designed to amplify a short (199 bp) or long (1561 bp) fragment of the listeriolysin (hly) gene. The short- and long-amplicon qPCR methods with and without propidium monoazide (PMA) treatment of the cells were tested for their ability to discriminate between viable (no heat) and heat-killed cells (90 °C, 10 min). The PMA-qPCR methods were subsequently used to assess the survival of L. monocytogenes during desiccation (33% RH, 15 °C) on stainless steel surfaces for ten days with and without prior biofilm formation. The long-amplicon qPCR method had a limit of quantification (LOQ) of 1.32 log CFU/reaction (efficiency 92%, R2 = 0.991), while the LOQ for the short-amplicon qPCR method was 1.44 log CFU/reaction (efficiency 102%, R2 = 0.991). PMA was essential for detection of viable cells, and the long-amplicon PMA-qPCR significantly (p < 0.05) reduced the signal from heat-killed cells compared to the short-amplicon method. L. monocytogenes survival during desiccation without biofilm formation was accurately enumerated with the long-amplicon PMA-qPCR method. However, when L. monocytogenes had formed biofilm prior to desiccation, the long-amplicon PMA-qPCR accurately measured the log fold inactivation but underestimated the number of viable cells even with use of an optimized DNA extraction method. This long-amplicon PMA-qPCR method can aid in the detection and enumeration of viable L. monocytogenes cells to further the understanding of its survival and persistence in food processing facilities. The developed method was demonstrated to work on both heat and desiccation treated cells and highlights the importance of amplicon size in viability-qPCR.

Synergistic Effect of Propidium Iodide and Small Molecule Antibiotics with the Antimicrobial Peptide Dendrimer G3KL against Gram-Negative Bacteria.

There is an urgent need to develop new antibiotics against multidrug-resistant bacteria. Many antimicrobial peptides (AMPs) are active against such bacteria and often act by destabilizing membranes, a mechanism that can also be used to permeabilize bacteria to other antibiotics, resulting in synergistic effects. We recently showed that G3KL, an AMP with a multibranched dendritic topology of the peptide chain, permeabilizes the inner and outer membranes of Gram-negative bacteria including multidrug-resistant strains, leading to efficient bacterial killing. Here, we show that permeabilization of the outer and inner membranes of Pseudomonas aeruginosa by G3KL, initially detected using the DNA-binding fluorogenic dye propidium iodide (PI), also leads to a synergistic effect between G3KL and PI in this bacterium. We also identify a synergistic effect between G3KL and six different antibiotics against the Gram-negative Klebsiella pneumoniae, against which G3KL is inactive.

Colicin N Mediates Apoptosis and Suppresses Integrin-Modulated Survival in Human Lung Cancer Cells.

The inherent limitations, including serious side-effects and drug resistance, of current chemotherapies necessitate the search for alternative treatments especially for lung cancer. Herein, the anticancer activity of colicin N, bacteria-produced antibiotic peptide, was investigated in various human lung cancer cells. After 24 h of treatment, colicin N at 5-15 µM selectively caused cytotoxicity detected by MTT assay in human lung cancer H460, H292 and H23 cells with no noticeable cell death in human dermal papilla DPCs cells. Flow cytometry analysis of annexin V-FITC/propidium iodide indicated that colicin N primarily induced apoptosis in human lung cancer cells. The activation of extrinsic apoptosis evidenced with the reduction of c-FLIP and caspase-8, as well as the modulation of intrinsic apoptosis signaling proteins including Bax and Mcl-1 were observed via Western blot analysis in lung cancer cells cultured with colicin N (10-15 µM) for 12 h. Moreover, 5-15 µM of colicin N down-regulated the expression of activated Akt (p-Akt) and its upstream survival molecules, integrin ß1 and αV in human lung cancer cells. Taken together, colicin N exhibits selective anticancer activity associated with suppression of integrin-modulated survival which potentiate the development of a novel therapy with high safety profile for treatment of human lung cancer.

Discrimination of viable and dead Vibrio parahaemolyticus subjected to low temperatures using Propidium Monoazide - Quantitative loop mediated isothermal amplification (PMA-qLAMP) and PMA-qPCR.

The aim of this study was to determine the effect of cold (4 °C) and subzero (-18 °C, -45 °C) temperatures on the occurrence time of membrane damage to provide Propidium Monoazide (PMA) penetration of Vibrio parahaemolyticus inoculated to the sea bass. Direct plate counting (DPC) and PMA-based quantitative loop-mediated isothermal amplification (qLAMP) and qPCR was utilized for discrimination of dead and live bacteria on the designated storage days (1, 3, 7, and 14). The optimum amount of PMA was 50 µM for inhibition of amplification derived from dead cells in spiked samples. The number of live V. parahaemolyticus was detectable at the end of the 14. day using PMA-qLAMP and PMA-qPCR at all the temperatures. On the 7th day, culturability has lost at any of the storage temperatures and DPCs at -18 °C and -45 °C revealed a difference of about 1 log10 CFU/ml between 1st and 3rd days. The same difference was also observed in PMA-qLAMP and PMA-qPCR on the same days (0.59-0.95 log10 CFU/ml). Subzero temperatures have the highest rate of viability while causing the fastest decrease in culturability in sample groups as a result of the higher level of transition to VBNC state. qLAMP and qPCR methods in the PMA-treated and nontreated groups on the storage days at all temperatures gave similar results (p > 0.05).

Efficacy of a rhamnolipid biosurfactant to inhibit Trichophyton rubrum in vitro and in a mice model of dermatophytosis.

Dermatophytic infections caused by Trichophyton rubrum are the most prevalent superficial mycoses worldwide. The present study was aimed at evaluating the anti-dermatophytic effect of a rhamnolipid biosurfactant (RL) produced by Pseudomonas aeruginosa SS14 (GenBank Accession no. KC866140) against T. rubrum in experimentally induced dermatophytosis in mice models. The purified RL could effectively suppress spore germination and hyphal proliferation of T. rubrum at 500 µg/mL. Ultramicroscopic observations involving SEM and AFM studies revealed severely altered hyphal morphology in the RL-treated mycelia. The membrane disruptive effect of RL was measured by the uptake of propidium iodide (PI) as visualized by CLSM. The absorbance of the RL-treated cell suspension at 260 nm showed concentration-dependant exudation of nucleic acid due to loss of cell membrane integrity. On evaluation of the therapeutic efficacy of RL on experimentally induced cutaneous dermatophytosis in mice models, we observed that the RL, when applied topically at a concentration of 500 µg/mL, was effective in completely curing dermatophytosis at the end of 21-day treatment period. The results were statistically similar to those obtained using the standard drug terbinafine as control. Apart from macroscopic observation, the results were confirmed by culture of skin scrapings and histopathological examination. The results are suggestive of the effectiveness of RL in control of dermatophytosis caused by T. rubrum.

Propidium monoazide-polymerase chain reaction for detection of residual periprosthetic joint infection in two-stage revision.

False negative culture results in periprosthetic joint infection (PJI) are not uncommon particularly when patients have received long term antibiotics. Polymerase chain reaction (PCR) has a lower specificity partly due to detection of residual DNA from dead bacteria. Propidium monoazide (PMA) prevents DNA from dead bacteria from being amplified during the PCR. This study aimed to determine the role of PMA in PCR for diagnosis of PJI. Clinical samples were tested by PCR with and without prior treatment with PMA and compared to conventional microbiological culture. The PCR assay included genus-specific primers for staphylococci and enterococci and species-specific primers for Cutibacterium acnes. The validated conditions of PMA treatment used in this study were 20 µM concentration and 5 and 10 min of dark incubation and photo-activation respectively. 202 periprosthetic tissues and explanted prostheses from 60 episodes in 58 patients undergoing revision arthroplasties for either PJI or non-infective causes were tested, by culture, PCR, and PMA-PCR. 14 of the 60 episodes satisfied the Musculoskeletal Infection Society (MSIS) criteria for PJI and 46 did not. Sensitivity of culture, PCR, and PMA-PCR were 50%, 71%, and 79% respectively. Specificities were 98%, 72%, and 89% respectively. All figures were calculated for episodes rather than samples. PMA-PCR enhanced both the specificity and the sensitivity of PCR. It has the potential to detect residual bacterial viability prior to reimplantation in the two-stage revision for PJI.