ddPCR titration of AAV vectors v2

This protocol describes ddPCR titration of AAV vectors. To see the full abstract and additional resources, visit https://www.addgene.org/protocols/aav-ddpcr-titration/. Sample Data: When analyzing data there should be a clear distinction between negative droplets (black) and positive droplets (blue). The no template control (NTC) should be close to zero (B08). At Addgene, runs with an NTC >5 are invalid. To reduce NTC values, we recommend wiping down all pipettes and equipment with 10% bleach prior to use and keeping all reagents and samples on ice or pre-chilled 96-well freezer blocks during use. In this protocol, a dilution series is prepared for each AAV sample and the 3 final dilutions are assayed. The samples that are assayed are diluted 2-fold serially therefore, the concentration obtained by ddPCR should decrease by a factor of 2 across the dilutions. In the example below, 2-fold serial dilutions of a sample were loaded in wells A04, A05 and A06. As shown in the image and table below, the concentration of positive droplets decreases by a factor of ~2. To increase the accuracy of the titer, calculate an average of several dilutions. For additional tips on AAV titering using ddPCR, read our blog post.

In vitro anthelmintic efficacy of Citrullus colocynthis (L.) Schrad on Haemonchus contortus

Ethno-veterinary medicinal studies associated with traditional uses of the flora of the Cholistan desert have shown that fruits of Citrullus colocynthis are used for the treatment of helminth infections. The present research was designed to evaluate the anthelmintic efficacy of C. colocynthis against H. contortus. The in vitro anthelmintic effects of aqueous-methanol and ethyl acetate fruit extracts of C. colocynthis against H. contortus were determined through egg hatch and adult motility assays. The effect of four serial dilutions of 25 mg/mL of each extract compared to levamisol (0.55 mg/mL) and oxfendazole (three serial dilutions of 25 µg/mL) were studied. Both ethyl acetate and aqueous-methanol extracts paralyzed all adult worms 4h and 8h post-exposure at a dose of 25 mg/mL each. In the egg hatch assay, about 83.67% and 80.67% of H. contortus eggs failed to hatch with the same dose (i.e. 25 mg/mL) of ethyl acetate and CAME extracts, respectively. The results of the present study strongly support fruit extracts of C. colocynthis as a promising alternative to synthetic drugs against H. contortus. These findings will lead to further in vivo studies to investigate the bio-availability of the active ingredients of the plant and the minimum non-lethal concentration required for treatment of haemonchosis in livestock. The anthelmintic effects of C. colocynthis might be attributed to the presence of phenolic acids.

Succussed Serial Dilutions in Water Carry Solute Information via Solute-Specific Water Structures–A Theory Based on Quantum Electrodynamics

Active ingredients are unlikely to be present in homeopathic dilutions that surpass the Avogadro limit. Yet responses of biological systems to these substances—chemically equivalent to water and indistinguishable from one another—are specific to the materials that are diluted away. This article addresses this challenging problem of homeopathy by identifying its underlying cause through a quantum electrodynamics-based “structural model” stated as: Succussed serial dilutions in water carry information about the solute via solute-specific water structures. The model is verifiable by our three-stranded set of experiments—nuclear magnetic resonance spectroscopy, anomalous dielectric dispersion, and atomic force microscopy. The results, some of which are presented here, directly or indirectly indicate that even extremely diluted solutions, devoid of any gross presence of the solutes, contain solute-reminiscent water structures. Apart from contributing to understanding high-dilution phenomena, these findings are expected to create an impact in the areas of medicine, pharmacopeia, and biology. Succussed aqueous dilutions acquire altered water structures with change of starting material: thus, their altered properties may be ascribed to these water structures, akin to allotropes of carbon. This theory justifies water structures as potential information carrier through succussed serial dilutions.

An Integrated Centrifugal Degassed PDMS-Based Microfluidic Device for Serial Dilution

We propose an integrated serial dilution generator utilizing centrifugal force with a degassed polydimethylsiloxane (PDMS) microfluidic device. Using gas-soluble PDMS as a centrifugal microfluidic device material, the sample can be dragged in any arbitrary direction using vacuum-driven force, as opposed to in a single direction, without adding further actuation components. The vacuum-driven force allows the device to avoid the formation of air bubbles and exhibit high tolerance in the surface condition. The device was then used for sample metering and sample transferring. In addition, centrifugal force was used for sample loading and sample mixing. In this study, a series of ten-fold serial dilutions ranging from 100 to 10−4 with about 8 μL in each chamber was achieved, while the serial dilution ratio and chamber volume could easily be altered by changing the geometrical designs of the device. As a proof of concept of our hybrid approach with the centrifugal and vacuum-driven forces, ten-fold serial dilutions of a cDNA (complementary DNA) sample were prepared using the device. Then, the diluted samples were collected by fine needles and subject to a quantitative polymerase chain reaction (qPCR), and the results were found to be in good agreement with those for samples prepared by manual pipetting.

Antimicrobial and antifungal activity of new fluorophenyl-containing 1,2,4-triazoles

The achievements of the world organic chemistry are most clearly represented by scientific publications that prove the promising nature of heterocyclic substances. The possibility of combining various pharmacophore fragments and 1,2,4-triazole in one molecule is quite popular. In 2018, the drug Trifuzol-NEO appeared on the veterinary market of Ukraine, which took its rightful place among synthetic immunomodulators for various groups of unproductive animals. So, further studies of new promising compounds among substituted 1,2,4-triazole, which can be used as objects for the creation of new original domestic antimicrobial and antifungal agents, remains relevant and has theoretical and practical significance. The aim of our work was to investigate the antimicrobial and antifungal activity of a number of new fluorophenyl-containing derivatives of 1,2,4-triazole and, in some cases, to trace the presence of certain patterns between structure and action. The sensitivity of new fluorophenyl-containing derivatives of 1,2.4-triazole was studied by the method of serial dilutions in accordance with the methodological recommendations. From the initial concentration of the new synthesized compounds of 1 mg/ml, a series of two-fold serial dilutions were prepared in Mueller–Hinton broth in a volume of 1 ml. Then, 0.1 ml of microbial curtain (106 m. c./ml) was added to each tube. MIC (MIC) was determined in the absence of visible growth in a test tube with the minimum concentration of the drug, the minimum bactericidal concentration (MBcK) – in the absence of growth on agar after inoculation from transparent tubes. Dimethyl sulfoxide was used as a solvent for the compounds in the studies. The research was carried out at the Department of Microbiology, Virology and Immunology of Zaporizhzhia State Medical University. Analyzing the results of studying the sensitivity of substances to Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, it should be noted that almost all compounds were active against the bacteria. It should be noted that a number of compounds were found to be the most active against Staphylococcus aureus, and 5-(2-fluorophenyl)-4-(((5-nitrofuran-2-yl)methylene)amino-4H-1,2,4-triazole-3-thiols generally exceeded several times the activity indices of the reference drug (MIC 1.95 μg/ml, MBcK 3.9 μg/ml) to Staphylococcus aureus. Most of the compounds were found to be quite active against Candida albicans. Among the corresponding 5-(2-fluorophenyl)-4-((aryl)ylidene)amino-1,2,4-triazole-3-thiols, the highest activity for Candida albicans exhibit 5-(2-fluorophenyl) -4-((4-bromophenyl)ylidene)amino-1,2,4-triazole-3-thiol and 5-(2-fluorophenyl)-4-((2,3-dimethoxyphenyl)ylidene)amino-1,2,4-triazole-3-thiol. It was found that most of the compounds exhibit a moderate antimicrobial and a fairly high antifungal effect. The most sensitive strain was S. aureus in relation to 5-(2-fluorophenyl)-4-(((5-nitrofurans-2-yl)methylene)amino-4H-1,2,4-triazole-3-thiol, Candida albicans proved to be very sensitive to 5-(2-fluorophenyl)-4-((4-bromophenyl)ylidene)amino-1,2,4-triazole-3-thiol and 5-(2-fluorophenyl)-4((2,3-dimethoxyphenyl)ylidene)amino-1,2,4-triazole-3-thiol.

Cytotoxicity of Cultured Canine Primary Hepatocytes Exposed to Itraconazole Is Decreased by Pre-treatment With Glutathione

Objective: To identify the effect of glutathione (GSH) on cell survival in a novel in vitro model of itraconazole (ITZ)-associated hepatotoxicity using canine primary hepatocytes.Sample: Commercially sourced, cryopreserved male dog (Beagle) primary hepatocytes from a single donor.Procedures: Using a sandwich culture technique, canine primary hepatocytes were exposed to serial dilutions of ITZ. Calcein AM, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), and neutral red were investigated as potential cell viability assays. Hepatocytes were then pre-incubated with GSH, exposed to serial dilutions of ITZ, and cell viability determined at 4 and 24 h post-ITZ exposure. Each condition was performed in technical triplicate and the effect of time, GSH concentration, and ITZ concentration on % cytotoxicity assessed using a multivariate linear regression model. Tukey's post-hoc test was used to detect individual differences.Results: The neutral red cell cytotoxicity assay was chosen based on its superior ability to detect dose-dependent changes in viability. Hepatocyte cytotoxicity significantly increased with ITZ concentration (P < 0.001) and time (P = 0.004) and significantly decreased with GSH treatment (P < 0.001).Conclusions and Clinical Relevance: This in vitro model demonstrates dose- and time-dependent ITZ-induced cytotoxicity, which is similar to clinical changes observed in canine patients and in in vivo rodent studies. Pre-treating with GSH is protective against in vitro cell death. These results suggest that GSH precursors may have a role in the management or prevention of ITZ-associated hepatotoxicity in dogs. Clinical trials are needed to evaluate their utility for this adverse drug reaction.

Clinical Value of CT for Differentiation between Ascites and Hemorrhage: An Experimental In-Vitro Study

Background: Abdominal trauma, leading to intra-abdominal bleeding, is a life-threatening condition that might need emergency surgical intervention. Sonography and Computed Tomography (CT) are most commonly used to detect free intra-abdominal fluid. This study investigates the accuracy of CT to distinguish between ascites and intra-abdominal hemorrhage. Methods: Ascites were collected during a clinical routine. Three serial dilutions, mixing ascites with whole blood samples of the patient and with two blood group identical donors, were prepared. Laboratory-chemical analysis and radiological evaluation using CT with measurement of average Hounsfield Units (HU) were performed. Results: Between ascites and whole blood as well as between ascites and the 1:1-ratio-samples, HU values differed significantly (p < 0.001). All further dilutions showed HU values with no significant difference compared to ascites (p ≥ 0.42). Whole blood showed significantly higher HU values than ascites and every step of the serial dilutions (p < 0.001). Measured HU values were also dependent on time and the exact point of measurement in the micro reaction vessels. Conclusions: In patients suffering from blunt abdominal trauma with preexisting ascites, HU values in CT imaging are not valid enough to exclude an acute hemorrhage.

Legionella antimicrobial sensitivity testing: comparison of microbroth dilution with BCYE and LASARUS solid media

Objectives There is a lack of international unification for AST methodology for Legionella pneumophila. Current literature contains multiple possible methods and this study compares each of them to determine methodological concordance. Methods Antibiotic susceptibility of 50 L. pneumophila strains was determined using broth microdilution (BMD), serial antimicrobial dilution in traditional buffered charcoal yeast extract (BCYE) agar (as well as comparison with gradient strip overlay on BCYE) and in a novel charcoal-free agar (LASARUS) for rifampicin, azithromycin, levofloxacin and doxycycline. Results The deviation of tested media relative to BMD highlighted the overall similarity of BMD and LASARUS across all antimicrobials tested (within one serial dilution). BCYE agar dilution showed an increased MIC of up to five serial dilutions relative to BMD, while MICs by gradient strip overlay on BCYE were elevated by two to three serial dilutions, with the exception of doxycycline, which was decreased by three serial dilutions relative to MIC values determined by BMD. The MIC range for azithromycin was wider than for other antimicrobials tested and found to be caused by the presence or absence of the lpeAB gene. Conclusions BMD-based antimicrobial susceptibility testing (AST) methodology should be the internationally agreed gold standard for Legionella spp. AST, as is common for other bacterial species. Traditional BCYE gave significantly elevated MIC results and its use should be discontinued for Legionella spp., while MIC determination using LASARUS solid medium gave results concordant (within one serial dilution) with BMD for all antimicrobials tested. To the best of our knowledge, this study is the first to identify the lpeAB gene in UK isolates.