Development of a capillary electrophoresis method for the screening of human urine for multiple drugs of abuse

A new capillary electrophoresis (CE) method was developed and validated for the screening of human urine for nineteen drugs of abuse. In order to achieve sufficient separation, the electrolyte composition was modified using beta-cyclodextrin (beta-CD) and organic solvents. To process each sample, a sequential injection-solid-phase extraction (SI-SPE) system was constructed. Using this device, matrix clean-up, extraction, and preconcentration of analytes were performed onto a C(18) cartridge. Optimal separation and detection were obtained using a background electrolyte consisting of 100mM phosphate adjusted to pH 6.0, with 20 mM beta-CD, 5% acetonitrile and 20% isopropanol. Electrokinetic injection was performed at 5 kV for 10s, separation voltage was 25 kV and column temperature was set to 25 degrees C. The separation was carried out in a 67.0 cm x 50 microm fused-silica capillary with UV detection at 214 nm. The combination of SI-SPE and sample stacking showed significant sensitivity enhancement with limits of detection in the range of 5-30 ng ml(-1). A validation study showed good reproducibility of both migration time (RSD=0.003-0.088%) and peak area (RSD=0.54-4.8%). Overall, this automated and miniaturized SI-SPE system provides a rapid, sensitive, and robust procedure for analysis; as well as minimizes sample and solvent consumption.     

Development of high-throughput screening system for osteogenic drugs using a cell-based sensor

To effectively treat osteoporosis and other bone-loss disorders, small compounds that potently induce bone formation are needed. The present study initially attempted to establish a monitoring system that could detect osteogenic differentiation easily, precisely, and noninvasively. For this purpose, we established pre-osteoblastic MC3T3E1 cells stably transfected with the GFP reporter gene driven by a 2.3 kb fragment of rat type I collagen promoter (Col1a1GFP-MC3T3E1). Among these cells, we selected a clone that fluoresced upon osteogenic stimulation by BMP2. The GFP fluorescence intensity corresponded well to the intensity of alkaline phosphatase (ALP) staining and to the level of osteocalcin (Oc) mRNA. Using this system, we screened natural and synthetic compound libraries and thus identified an isoflavone derivative, glabrisoflavone (GI). GI induced ALP staining and Oc mRNA in a dose-dependent manner. The Col1a1GFP-MC3T3E1 system may be useful for identifying novel osteogenic drugs.     

Development of a dot-blot assay for screening monoclonal antibodies to low-molecular-mass drugs

Dot-blot is a versatile and simple analysis to perform. We adapted this method as a simple identity test for monoclonal antibodies to a number of small compounds: three transplant drugs, an anticonvulsant, a steroid, an anticancer drug, and an antibiotic. Immunology-based identity tests using low-molecular-mass organic compounds have historically been a challenge to develop. We modified the traditional dot-blot assay to serve as an identity test for monoclonal antibodies to carbamazepine, sirolimus, tacrolimus, cyclosporine, cortisol, methotrexate, and gentamicin. The primary obstacle was the immobilization of these organic compounds on nitrocellulose as nitrocellulose is also soluble in most of the organic solvents in which the compounds are soluble. We evaluated different membranes, solvents, and chemical forms of these organic compounds to overcome this challenge. A number of incubation and washing solutions were also investigated. By varying the chemical form, concentration, and incubation conditions, a set of effective and reproducible identity tests were developed for these monoclonal antibodies.     

An invertebrate hyperglycemic model for the identification of anti-diabetic drugs

The number of individuals diagnosed with type 2 diabetes mellitus, which is caused by insulin resistance and/or abnormal insulin secretion, is increasing worldwide, creating a strong demand for the development of more effective anti-diabetic drugs. However, animal-based screening for anti-diabetic compounds requires sacrifice of a large number of diabetic animals, which presents issues in terms of animal welfare. Here, we established a method for evaluating the anti-diabetic effects of compounds using an invertebrate animal, the silkworm, Bombyx mori. Sugar levels in silkworm hemolymph increased immediately after feeding silkworms a high glucose-containing diet, resulting in impaired growth. Human insulin and 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), an AMP-activated protein kinase (AMPK) activator, decreased the hemolymph sugar levels of the hyperglycemic silkworms and restored growth. Treatment of the isolated fat body with human insulin in an in vitro culture system increased total sugar in the fat body and stimulated Akt phosphorylation. These responses were inhibited by wortmannin, an inhibitor of phosphoinositide 3 kinase. Moreover, AICAR stimulated AMPK phosphorylation in the silkworm fat body. Administration of aminoguanidine, a Maillard reaction inhibitor, repressed the accumulation of Maillard reaction products (advanced glycation end-products; AGEs) in the hyperglycemic silkworms and restored growth, suggesting that the growth defect of hyperglycemic silkworms is caused by AGE accumulation in the hemolymph. Furthermore, we identified galactose as a hypoglycemic compound in jiou, an herbal medicine for diabetes, by monitoring its hypoglycemic activity in hyperglycemic silkworms. These results suggest that the hyperglycemic silkworm model is useful for identifying anti-diabetic drugs that show therapeutic effects in mammals.     

Development of a simplified method for subclass isotyping and screening monoclonal antibodies

A simplified dot-blot procedure for screening and subclass isotyping of monoclonal antibodies is described in which only 0.5-50 ng of antigen and 1 microliter of antibody are needed to perform the test. The results on the nitrocellulose membrane can be stored indefinitely for future reference. This method is less expensive, uses smaller quantities of antigen and antibody, and is faster than presently used enzyme-linked immunosorbent assay techniques or other dot-blot methods. Monoclonal antibodies against Mycoplasma gallisepticum were screened and isotyped using this technique.     

Development of a method for screening short-lived proteins using green fluorescent protein

We have developed a screening technology for the identification of short-lived proteins. A green fluorescent protein (GFP)-fusion cDNA library was generated for monitoring degradation kinetics. Cells expressing a subset of the GFP-cDNA expression library were screened to recover those in which the fluorescence signal diminished rapidly when protein synthesis was inhibited. Thirty clones that met the screening criteria were characterized individually. Twenty-three (73%) proved to have a half-life of 4 hours or less.     

Development of a high-content screening method for chemicals modulating DNA damage response

The cellular response to DNA damage is emerging as a promising target for cancer therapy. In the present study, the authors exploited the relationship between the level of the phosphorylated form of histone H2AX (γH2AX) and the extent of DNA damage and developed a quantitative, cell-based, high-content screening system for measuring the DNA damage response (DDR). In this system, the authors quantified the level of γH2AX by measuring DNA damage-induced γH2AX nuclear foci using an automated cell imager. They found that the total area of γH2AX foci per cell exhibited a good correlation with the concentration of DNA damage-inducing agents, including etoposide. The effects of 2 well-known inhibitors of DNA damage could be quantified using this system, suggesting the suitability of the γH2AX-foci quantification method for large-scale screening applications. This was confirmed by using this method to screen a chemical library; the resulting "hits" included compounds that inhibited early signaling events in DDR, as well those that inhibited subsequent DNA damage repair processes. Overall, this γH2AX foci-measuring system may be an effective screening method for identifying DNA damage response inhibitors that could eventually be used to develop novel anticancer drugs.     

Assessment of drugs against Cryptosporidium parvum using a simple in vitro screening method

A rapid semi-quantitative screening method was devised for assessing the anticryptosporidial and cytotoxic effects of putative chemotherapeutic compounds. The method is suitable as an initial rapid screening procedure from which compounds demonstrating anticryptosporidial activity can be identified for further analysis. It has the advantages of speed, low cost and concurrent assessment of anticryptosporidial and cytotoxic effects and allows accurate determination of minimum lethal concentrations. Of the 71 compounds screened, six completely inhibited cryptosporidial growth at 1 microM (monensin, salinomycin, alborixin, lasalocid, trifluralin and nicarbazin) and a further eight showed significant anticryptosporidial activity at 1 or 20 microM (halquinol, bleomycin, suramin, mitomycin, doxycycline hydrochloride, toltrazuril, chloroquine phosphate and teniposide). Twelve compounds were found to have some degree of cytotoxicity at 1 microM and a further 12 at 20 microM.     

Development and validation of isomer specific RP-HPLC method for quantification of cefpodoxime proxetil

The present work explains the development and validation of a simple and reliable isomer specific liquid chromatographic method for the quantitative determination of cefpodoxime proxetil (CP) in rat in situ intestinal perfusate samples. Chromatography was carried out by reversed-phase technique on a C-18 column with a mobile phase composed of 20 mM ammonium acetate buffer (pH 5.0) and acetonitrile in the ratio of 62:38 pumped at a flow-rate of 1 ml/min. The detection was carried out at 235 nm and a column temperature of 30 degrees C. The method was evaluated for the various validation parameters, such as linearity, accuracy, precision, LOD, LOQ, specificity, selectivity, and sample stability. The results of intra- and inter-day validation (n = 3) showed the method to be efficient and the same was applied in an in situ permeability study conducted for CP in rats.     

Development and validation of a RP-HPLC method for quantification of isoflavone aglycones in hydrolyzed soy dry extracts

Isoflavones are widely used as an alternative treatment to hormone replacement therapy and also for prevention of several chronic diseases, including cancers. Genistein, daidzein and glycitein are the most abundant isoflavone aglycones found in soy extracts, where they also occur as glycosides. This paper describes the development and validation of an isocratic reversed-phase HPLC (RP-HPLC) method for the analysis of isoflavone aglycones, released after acid hydrolysis of soy dry extracts, used as pharmaceutical raw material. The quantification was carried out in a C(18) endcapped column, using a mobile phase composed of 0.1% acetic acid and methanol (52:48), at a flow rate of 1.0 ml/min and diode array detection (DAD) at 254 nm. The method showed to be linear (r(2)>0.99), precise (R.S.D.<2%), accurate (recovery of 98.88% for daidzein and 98.12% for genistein), robust and specific.     

Development of a sensitive bioassay method for quorum sensing inhibitor screening using a recombinantAgrobacterium tumefaciens

Acylhomoserine lactones (AHLs) are known to be the triggering molecules in the quorum sensing mechanism of many gram-negative bacteria. In order to detect AHL inhibitors that are potential biofilm inhibitors, a convenient and sensitive bioassay was developed based on the β-galactosidase activity (β-GAL) of a recombinantAgrobacterium tumefaciens strain. A series of commercially available AHLs were tested for inducing β-GAL at varying concentrations in agar-plate and liquid cultures of the reporter strain. All AHLs tested exhibited a concentration-dependent induction, and octanoyl homoserine lactone (OHL) showed the highest sensitivity with a detection limit of 0.1 nM in the liquid culture assay. When fimbrolide, a known quorum sensing inhibitor, was added, induction of β-GAL by OHL was repressed. The repression at a constant OHL concentration was dependent on the fimbrolide concentration with the detection limit below 1 ppm, indicating that this assay is a sensitive method for screening AHL inhibitors.     

Tartrazine: a potentially hazardous dye in Canadian drugs.

The literature was reviewed to determine the incidence of idiosyncratic reactions to tartrazine. From 4% to 14% of individuals with asthma or allergies or both and from 7% to 20% of persons who are sensitive to acetylsalicylic acid may react to this dye. The mechanism of such reactions is unknown. Pharmaceutical manufacturers and distributors were surveyed and a list was prepared of approximately 450 Canadian pharmaceuticals that contain tartrazine. The 53 pharmaceutical and manufacturers and distributors whose drug products do not contain this dye were also listed. It is recommended that information concerning the tartrazine content of drugs be included on package labels.     

Development of a high-throughput screening method for recombinant Escherichia coli with intracellular dextransucrase activity

To efficiently engineer intracellular dextransucrase (DSase) expression in Escherichia coli, a high-throughput screening method was developed based on the polymer-forming activity of the enzyme. Recombinant E. coli containing the Leuconostoc citreum DSase (LcDS) gene was grown on Luria-Bertani agar plates, containing 2% sucrose, at 37°C for 8 h. The plates were then evenly overlaid with 0.6% soft agar, containing 1.2 mg/ml D-cycloserine, and incubated at 30°C to allow gradual cell disruption until a dextran polymer grew through the overlaid layer. A significant correlation between dextran size and enzyme activity was established and applied for screening truncated mutants with LcDS activity.     

Development and application of a novel and effective screening method for aerobic denitrifying bacteria

Herein we describe a novel and effective screening method for aerobic denitrifying bacteria. For this procedure, we utilized KCN to inhibit the electron transference from Cytaa3 to oxygen in the bacteria respiratory chain. We employed a 3-h aeration operation cycle and intermittent rotations. The resultant bacterial suspensions were plated on a KCN-screening medium and incubated aerobically. Single colonies were selected and incubated in an aerobic culture medium. Culture nitrate and nitrite levels were determined over time, and ultimately four bacterial strains that performed denitrifying under aerobic conditions were identified by this method. Of these, strain Y2-1-1 demonstrated the best aerobic denitrifying ability. In a 5-day test, the NO3--N of the aerobic culture medium was reduced from 282.0+/-8.3 mg L(-1) to 149.2+/-17.1 mg L(-1), with little nitrite or N2O production. The morphological, physiological and biochemical characteristics and the 16S rRNA gene sequence homology comparison data for this strain were consistent with the classification of the genus Pseudomonas. We named this strain Pseudomonas sp. Y2-1-1.     

A mechanism-based, solution-phase method for screening combinatorial mixtures of potential platinum anticancer drugs

 We report a new, mechanism-based approach to the screening of pools of potential platinum antitumor drugs. A platinum complex of L-lysine, [Pt(Lys)Cl₂] or Kplatin, was selected from mixtures of platinum-amino acid compounds based on the ability of its DNA adducts to bind HMG1 in a gel mobility shift assay. Kplatin, unlike most other platinum antitumor drug candidates, is an (N,O)-chelated complex which binds DNA forming two isomeric 1,2-d(GpG) intrastrand DNA cross-links. Kplatin-modified DNA is specifically recognized by HMG1, HMG1 domain B, and testis-specific HMG, all of which bind to the major cisplatin-DNA adducts. Kplatin is toxic towards the human tumor cell lines HeLa and KM12 with LC₅₀ values of 59.2±7.8 μM and 74 μM, respectively. Received: 16 July 1997 / Accepted 31 October 1997     

Development of a biosensor assessing SARS-CoV-2 main protease proteolytic activity in living cells for antiviral drugs screening

Highlights:
The biosensor reported in our study can monitor SARS-CoV-2 M^pro activity in living cells instead of in vitro solutions.
The biosensor reported in our study is sensitive and easy to operate.
It is suitable for high-throughput screening.
It has the potential to be used in small animal models.     

Development of a sensitive screening method for selecting monoclonal antibodies to be internalized by cells

Antibody–drug conjugates (ADCs), drugs developed by conjugation of an anticancer agent to a monoclonal antibody (mAb), have lately attracted attention in cancer therapy because ADCs can directly bind cancer cells and kill them. Although mAbs for ADCs must be internalized by the target cells, few methods are available for screening mAbs for their ability to be internalized by cells. We have developed a recombinant protein, termed DT3C, which consists of diphtheria toxin (DT) lacking the receptor-binding domain but containing the C1, C2, and C3 domains of Streptococcus protein G (3C). When a mAb–DT3C conjugate, which functions in vitro like an ADC, reduces the viability of cancer cells, the mAb being tested must have been internalized by the target cells. DT3C can thus be a tool to identify efficiently and easily mAbs that can be internalized by cells, thereby enhancing the development of promising ADCs.     

Development of a screening method for the indentification of a novelSaccharomyces cerevisiae mutant over-expressingTrichoderma reesei cellobiohydrolase II

In a previous study we showed that the fusion of the cellulose-binding domain (CBD2) fromTrichoderma reesei cellobiohydrolase II to a β-glucosidase (BGL1) enzyme fromSaccharomycopsis fibuligera significantly hindered its expression and secretion inSaccharomyces cerevisiae. This suggests that the possible low secretion of heterologous cellulolytic enzymes inS. cerevisiae could be attributed to the presence of a cellulose-binding domain (CBD) in these enzymes. The aim of this study was to increase the extracellular production of the chimeric CBD2-BGL1 enzyme (designated CBGL1) inS. cerevisiae. To achieve this, CBGL1 was used as a reporter enzyme for screening mutagenisedS. cerevisiae strains with increased ability to secrete CBD-associated enzymes such as cellulolytic enzymes. A mutant strain ofS. cerevisie, WM91-CBGL1, which exhibited up to 200 U L^?1 of total activity, was isolated. Such activity was approximately threefold more than that of the parental host strain. Seventy-five per cent of the activity was detected in the extracellular medium. The mutant strain transformed with theT. resei CBH2 gene produced up to threefold more cellobiohydrolase enzyme than the parental strain, but with 50% of the total activity retained intracellularly. The cellobiohydrolase enzymes from the parent and mutant strains were partially purified and the characteristic properties analysed.