Incorporation of cadmium into proteins in a cadmium tolerant fungi

Aspergillus carbonarius and a strain of Penicillium, a cadmium tolerant fungi, are able to metabolize cadmium chloride up to 2% (w/v). Their amino acids analysis on cadmium free and cadmium chloride containing media indicated certain disorders in their metabolic activities. Cystathionine was only detected in both fungi in the presence of cadmium chloride. However, cadmium was incorporated into several types of low and high molecular weight proteins. The amino acids hydrolyzates of a cadmium containing protein are characterized by the presence of high levels of sulfur amino acids; cysteine and methionine. Ethylasparagine was detected in the hydrolyzate of that cadmium containing protein as well.     

Mobilization of selenium by a selenium-dependent bacterium

A selenium-dependent bacterium, Bacillus sp., failed to grow on selenium-free media. However, it is able to grow at high concentrations of sodium selenite containing media up to 3% (w/v). It accumulated extraordinary high quantities of selenium, 432 ppm/mL. The bacterium generated the transformation of inorganic selenium into volatile selenium form(s) into the atmosphere. The biological release of volatile selenium basically depended on several factors: incubation temperature, pH, incubation periods, and substrate concentration. Maximal quantity of the volatile selenium form was obtained at 30 degrees C, pH 7, and 1% (w/v) sodium selenite.     

Novel complex Re-Arrangement of ARG1 commonly shared by unrelated patients with Hyperargininemia

Background

Hyperargininemia is a very rare progressive neurometabolic disorder caused by deficiency of hepatic cytosolic arginase I, resulting from mutations in the ARG1 gene. Until now, some mutations were reported worldwide and none of them were of Southeast Asian origins. Furthermore, most reported mutations were point mutations and a few others deletions or insertions.

Objective

This study aims at identifying the disease-causing mutation in the ARG1 gene of Malaysian patients with hyperargininemia.

Methodology

We employed a series of PCR amplifications and direct sequencing in order to identify the mutation. We subsequently used quantitative real-time PCR to determine the copy number of the exons flanking the mutation. We blasted our sequencing data with that of the reference sequence in the NCBI in order to obtain positional insights of the mutation.

Results

We found a novel complex re-arrangement involving insertion, inversion and gross deletion of ARG1 (designated g.insIVS1 + 1899GTTTTATCAT;g.invIVS1 + 1933_ + 1953;g.delIVS1 + 1954_IVS2 + 914;c.del116_188;p.Pro20SerfsX4) commonly shared by 5 patients with hyperargininemia, each originating from different family. None of the affected families share known relationship with each other, although four of the five patients were known to have first-cousin consanguineous parents.

Conclusion

This is the first report of complex re-arrangement in the ARG1. Further analyses showing that the patients have shared the same geographic origin within the northeastern part of Malaysia prompted us to suggest a simple molecular screening of hyperargininemia within related ethnicities using a long-range PCR.     

Mixed Herbs Drugs: Inhibitory Effect on Growth of the Endogenous Mycoflora and Aflatoxin Production

Twenty commercial mixed herbal drugs were examined for mycological profile. Aspergillus species were the predominant fungi found in the drugs. Other fungi harboured in the drugs with less frequency were Paecilomyces species, Eurotium species, Monascus species, Acremonium species, Penicillium species, Cladosporium species, Scopulariopsis species, Phialophora species and Fonseceae species. Fungal count was between 1.0 log₁₀ CFU and 2.4 log₁₀ CFU per gram of sample. When the drugs were incubated in 85% humidity at 25°C, fungal colonies grew on only two of the drugs. The mixed herbal drugs were extracted with water and the extracts were used to grow Aspergillus parasiticus. All extracts reduced aflatoxin B₁ and aflatoxin G₁ production by 62–97%. All but two of the extracts reduced aflatoxin B₂ and aflatoxin G₂ production by 39–95%. It can be concluded that the commercial powdered mixed herbal drugs contained low number of endogenous fungi, and these drugs are inhibitory to the growth of its endogenous fungi and aflatoxins production by aflatoxigenic fungi.     

Simple, rapid and sensitive assay method for simultaneous quantification of urinary nicotine and cotinine using gas chromatography-mass spectrometry

Nicotine is a major addictive compound in cigarette. Its smoke is rapidly and extensively metabolized to several metabolites in human. Cotinine as a major metabolite of nicotine is commonly used as a biomarker to determine active and passive smokers. Cotinine has a longer half-life ( approximately 20 h) compared to nicotine ( approximately 2h). A simple, sensitive, rapid and high throughput GC-MS method was developed for simultaneous quantification of urinary nicotine and cotinine in passive and active smokers. In the sample preparation method, the analytes and internal standard were first basified and followed by liquid-liquid extraction. Upon completion, anhydrous sodium sulphate was added to the solvent mixture to trap moistures. The clear extract obtained was directly injected into GC-MS, operating under selective ion monitoring (SIM) mode. Calibration curves in the range of 0.5-5000 ng/mL of the analytes in urine matrix were established with linear correlation coefficients (r(2)) greater than 0.997. The limit of detection for both nicotine and cotinine were 0.20 ng/mL. The mean recoveries for nicotine and cotinine were 93.0 and 100.4%, respectively. The within- and between-assay accuracies were between 2.1 and 7.9% for nicotine and between 0.7 and 11.1% for cotinine. Within- and between-assay precisions of 3.3-9.5% for nicotine and 3.4-9.8% for cotinine were also achieved. The method can be used in routine assessment and monitoring of active smoking and exposure to environmental tobacco smoke. The applicability of the assay was demonstrated in a small-scale comparison study between smokers and non-smokers.     

3D Mapping of Safe and Danger Zones in the Maxilla and Mandible for the Placement of Intermaxillary Fixation Screws

Intermaxillary (IMF) screws feature several advantages over other devices used for intermaxillary fixation, but using cone beam computed tomography (CBCT) scans to determine the safe and danger zones to place these devices for all patients can be expensive. This study aimed to determine the optimal interradicular and buccopalatal/buccolingual spaces for IMF screw placement in the maxilla and mandible. The CBCT volumetric data of 193 patients was used to generate transaxial slices between the second molar on the right to the second molar on the left in both arches. The mean interradicular and buccopalatal/buccolingual distances and standard deviation values were obtained at heights of 2, 5, 8 and 11 mm from the alveolar bone crest. An IMF screw with a diameter of 1.0 mm and length of 7 mm can be placed distal to the canines (2 - 11 mm from the alveolar crest) and less than 8 mm between the molars in the maxilla. In the mandible, the safest position is distal to the first premolar (more than 5 mm) and distal to the second premolar (more than 2 mm). There was a significant difference (p<0.05) between the right and left quadrants. The colour coding 3D template showed the safe and danger zones based on the mesiodistal, buccopalatal and buccolingual distances in the maxilla and mandible.The safest sites for IMF screw insertion in the maxilla were between the canines and first premolars and between the first and second molars. In the mandible, the safest sites were between the first and second premolars and between the second premolar and first molar. However, the IMF screw should not exceed 1.0 mm in diameter and 7 mm in length.     

A facile chemo-, regio- and stereoselective synthesis and cholinesterase inhibitory activity of spirooxindole–pyrrolizine–piperidine hybrids

A series of novel hybrid spiro heterocycles comprising pyrrolizine, spiroxindole and piperidine moieties was synthesized chemo-, regio- and stereoselectively in good yields from 1,3-dipolar cycloaddition reaction of a series of 1-acryloyl-3,5-bisarylmethylidenepiperidin-4-ones with azomethine ylides generated in situ from 5-choloroisatin and l-proline in methanol. These cycloadducts displayed significant cholinesterase inhibitory activity. Among the compounds screened, 8g and 8e, showed maximum inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinestrase (BChE) with IC₅₀ values of 3.33 and 3.13 μM, respectively.     

Efficiency of New Dose Escalation Designs in Dose-Finding Phase I Trials of Molecularly Targeted Agents

Background

Statistical simulations have consistently demonstrated that new dose-escalation designs such as accelerated titration design (ATD) and continual reassessment method (CRM)-type designs outperform the standard “3+3” design in phase I cancer clinical trials.

Methods

We evaluated the actual efficiency of different dose escalation methods employed in first-in-human phase I clinical trials of targeted agents administered as single agents published over the last decade.

Results

Forty-nine per cent of the 84 retrieved trials used the standard “3+3” design. Newer designs used included ATD in 42%, modified CRM [mCRM] in 7%, and pharmacologically guided dose escalation in 1%. The median numbers of dose levels explored in trials using “3+3”, ATD and mCRM designs were 6, 8 and 10, respectively. More strikingly, the mean MTD to starting dose ratio appeared to be at least twice as high for trials using mCRM or ATD designs as for trials using a standard “3+3” design. Despite this, the mean number of patients exposed to a dose below the MTD was similar in trials using “3+3”, ATD and mCRM designs.

Conclusion

Our results support a more extensive implementation of innovative dose escalation designs such as mCRM and ATD in phase I cancer clinical trials of molecularly targeted agents.