Automatic microscopic detection of mycobacteria in sputum: a proof-of-concept.

The laboratory diagnosis of lung mycobacterioses including tuberculosis comprises the microscopic examination of sputum smear after appropriate staining such as Ziehl-Neelsen staining to observe acid-fast bacilli. This standard procedure is operator-dependant and its sensitivity depends on the duration of observation. We developed and evaluated an operator-independent microscopic examination of sputum smears for the automated detection and enumeration of acid-fast bacilli using a ZEISS Axio Scan.Z1 microscope. The sensitivity, specificity, positive predictive value, negative predictive values and accuracy were calculated using standard formulations by comparison with standard microscopic examination. After in-house parameterization of the automatic microscope and counting software, the limit of detection evaluated by seeding negative sputa with Mycobacterium bovis BCG or Mycobacterium tuberculosis H37Rv (100-105 bacilli/mL) was of 102 bacilli/mL of sputum with a 100% positivity rate. Then, the evaluation of 93 sputum specimens including 34 smear-positive and 59 smear-negative specimens yielded a sensitivity of 97.06% [84.67-99.93%], a specificity of 86.44% [73.01-92.78%]. Up to 100 smear slides could be stocked for reading in the microscope magazine and results are exportable into the laboratory information system. Based on these preliminary results, we are implanting this automatic protocol in the routine workflow so that only smears detected positive by automatic microscopy are confirmed by standard microscopic examination.

The Fauna and Active Season of Mosquitoes in West of Fars Province, Southwest of Iran.

Culicidae are highly important for public health as they can be vectors of diseases and are responsible for a wide spectrum of infections. Five collection sites were selected randomly with regards to existing facilities in Firouzabad County. For collecting larvae and total catch for adult mosquitoes, sampling was carried out by dipping technique for collecting larvae and total catch for adult mosquitoes. A total of 689 adults and 1313 larvae of Culicidae were collected, of which 3 genera and 6 species of Culicidae were recognized, namely, Anopheles superpictus, Anopheles d’thali, Culex sinaiticus, Culex theileri, Culex mimeticus, and Culiseta longiareolata. Cx. theileri was the most frequent Culicidae collected at Firouzabad, with a total of 613 and 247 larval and adult specimens, respectively. The highest number of mosquitoes was collected in June (31.1%) and the lowest in May (3.4%). The mean temperatures in June and May were 31.3˚C and 28.2˚C, respectively. We found some vectors that are of medical and veterinary importance; our results could be applied in vector control programs that aim at eradication or control of mosquitoes in this area.

In Silico and In Vitro Evaluation of Cytotoxic Activities of Farnesiferol C and Microlobin on MCF-7, HeLa and KYSE Cell Lines.

Background: Cancer is one of the leading causes of death worldwide. Despite certain advances in cancer therapy, still there is considerable demand for developing efficient therapeutic agents. Nowadays, there is a rising interest in the use of natural-based anti-cancer drugs. In this study, the cytotoxicity of farnesiferol C and microlobin isolated from Ferula szowitsiana was investigated against MCF-7, HeLa and KYSE cancer cell lines. In addition, the mechanism of binding of these compounds to apoptotic proteins (Bax, Bak and Bcl-2) was analyzed by an in silico method. Materials and methods: We used MTT assay in order to assess the cytotoxicity of compounds against cancer cell lines. For in silico study, the AutoDock 4 was adopted. Results and discussion: According to the in vitro findings, in general, farnesiferol C showed significant cytotoxicity at higher concentrations (>50 µM) following 48 and 72 h incubation with the selected cancer cells; however, microlobin exhibited almost no activity at concentrations up to 100 µM. The in silico results revealed that both compounds could bind to Bax more efficiently rather than to Bcl-2 or Bak proteins. Conclusion: The results obtained by our preliminary in vitro and in silico studies suggest that these compounds might induce apoptosis through Bax activation; however further studies, either in vitro or in vivo are needed to clarify these activities.

Antituberculosis agents IV: in vitro antimycobacterial activity and cytotoxicity of N-piperazinyl quinolone derivatives containing 2-thienyl and 2-furyl moiety.

A series of N-[2-(2-furyl)-2-oxoethyl], N-[2-(2-furyl)-2-oxyiminoethyl], N-[2-oxo-2-(2-thienyl)ethyl] and N-[2-oxyimino-2-(2-thienyl)ethyl] piperazinyl quinolones (1a-h; 2a-h) were evaluated for antituberculosis activity against M. tuberculosis H37Rv using the BACTEC 460 radiometric system and BACTEC 12B medium. Our results indicated that compounds 1a, 1e and 1g were efficient antimycobacterial agents showing MIC values ranging from 0.78 to 6.25 microg/ml. In general, ciprofloxacin derivatives were more active than norfloxacin derivatives and the oxime analogues were less active than corresponding ketones. Active compounds (1a, 1e and 1g) were also screened by serial dilution to assess toxicity to VERO cell line. The cytotoxicity of tested compounds indicated that compound 1a was the less toxic compound (IC50 > 62.5 microg/ml). This compound was tested for efficacy in vitro in TB-infected macrophage model (EC90 = 3.25 microg/ml).

Antituberculosis agents. IX. In vitro antimycobacterial activity of N-(2-phenyl-2-oxoethyl) and N-[2-(4-fluorophenyl)-2-oxoethyl]ciprofloxacin derivatives against some drug-resistant strains of Mycobacterium tuberculosis and Mycobacterium avium isolates.

The in vitro antimycobacterial activity of two ciprofloxacin analogues (2a and 2b) containing 2-phenyl-2-axoethyl and 2-(4-fluorophenyl)-2-axoethyl groups attached to N-4 position of piperazin ring, was evaluated against M. tuberculosis strains resistant to Isoniazid (MIC 2a and 2b = 3.13 micrograms/ml), Ethambutol (MIC 2a and 2b = 1.56 micrograms/ml), Rifampin (MIC 2a and 2b = 1.56 micrograms/ml), Kanamycin (MIC 2a and 2b = 1.56 micrograms/ml) and ciprofloxacin (MIC 2a and 2b > 25 micrograms/ml). Furthermore, the minimum bactericidal concentration (MBC) of 2a and 2b was determined against M. tuberculosis H37Rv (MBC2a = 6.25 and 2b = 25 micrograms/ml) and strains resistant to isoniazid (MBCs > 25 micrograms/ml) and rifampin (MBC2a = 3.13 and 2b = 6.25 micrograms/ml). Also, in this study the activity of 2a and 2b was determined against a strain of M. avium (%Inhibition 2a = 89 and 2b = 95%). Expanded primary screening was conducted for 2b (having MIC < 6.25 micrograms/ml) against five clinical isolates of M. avium using the MABA and BACTEC 460 systems (MIC = 2-4 micrograms/ml). The significant anti-Mycobacterium avium activity of 2b suggested further M. avium assays and so, 2b was then retested at lower concentrations against 30 strains of M. avium including five strains resistant to claritromycin (MIC = 2-16 micrograms/ml).

Antituberculosis agents. VII. Synthesis and in vitro evaluation of antimycobacterial activity and cytotoxicity of some N-piperazinyl quinolone derivatives.

A series of N-[2-(2,4-dichlorophenyl)-2-oxoethyl] and N-[2-aryl-2-benzyloxyimino ethyl]piperazinyl quinolones (5-13) have been prepared as part of a study to examine the relationship between structural modification at 7-position and activity against Mycobacterium tuberculosis. Primary screening was conducted at concentration of 6.25 micrograms/ml against M. tuberculosis H37Rv using BACTEC 460 radiometric system and BACTEC 12B medium. The actual minimum inhibitory concentrations (MIC) were determined for compounds demonstrating at least 90% inhibition in the primary screening. The results demonstrate that substitution of phenyl ring with 4- fluoro, 2, 4-difluoro and 2,4-dichloro groups resulted in variable inhibition percentage (Inh% = 7-101). Despite the significant antituberculosis activity of ciprofloxacin and norfloxacin derivatives containing 2-(2,4-dichlorophenyl)-2-oxoethyl moiety (MIC = 0.39 & 6.25 micrograms/ml), compounds with 2-aryl-2-(hydroxyimino)ethyl, 2-aryl-2-(benzyloxyimino)ethyl and 2-aryl-2-(4-chlorobenzyloxyimino)ethyl did not show any activity (MIC > 6.25 micrograms/ml, Inh% = -7 to 75). Active compounds were also screened by serial dilution to assess toxicity to a VERO cell line. While the most active compound 5a was not soluble in tissue culture media, compound 5b showed IC50 = 5.3 micrograms/ml.

Antituberculosis agents. III. In vitro evaluation of antimycobacterial activity and cytotoxicity of some N-piperazinyl quinolone derivatives.

A series of N-[2-oxo-2-(4-substitutedphenyl)ethyl]piperazinyl quinolones(1a-e,2a-e and 3a-c) and N-[2-hydroxyimino-2-(4-substitutedphenyl)ethyl]piperazinyl quinolones(1f-j,2f-j and 3d-f) were evaluated for antituberculosis activity against Mycobacterium tuberculosis H37R, using the BACTEC 460 radiometric system and BACTEC 12B medium. Active compounds were also screened by serial dilution to assess toxicity to a VERO cell line. Nine compounds were efficient antimycobacterial agents showing MIC values ranging from 0.78 to 6.25 micrograms/ml. Generally, ciprofloxacin derivatives were more active than norfloxacin and enoxacin derivatives and the oxime analogues were less active than corresponding ketones. The most selective and less toxic compound 1a was tested for efficacy in vitro in TB-infected macrophage model (EC90 = 3.68 micrograms/ml, EC99 = 9.18 micrograms/ml).

Antituberculosis agents. VI. Activity of a new ciprofloxacin derivative against Mycobacterium avium and some drug-resistant strains of Mycobacterium tuberculosis.

The in vitro antimycobacterial activity of a new quinolone derivative, 1a containing 2-(2-furyl)-2-oxoethyl group at N-4 position of piperazine ring of Ciprofloxacin was tested for efficacy in vitro in TB-infected macrophage model (EC90 = 3.25 micrograms/ml and EC99 > 12.5 micrograms/ml). The MIC values of 1a were determined against M. tuberculosis strains resistant to Isoniazid (MIC = 1.56 micrograms/ml), Rifampin (MIC = 1.56 micrograms/ml), Ethambutol (MIC = 0.78 microgram/ml), Kanamycin (MIC = 0.78 microgram/ml) and Ciprofloxacin (MIC > 25 micrograms/ml). Furthermore, the MIC and selectivity index (SI) of 1a were evaluated against M. avium. Also, in this study the minimum bactericidal concentration (MBC) of 1a was determined against M. tuberculosis H37Rv (MBC = 6.25 micrograms/ml) and strain resistant to Rifampim (MBC = 25 micrograms/ml) and Isoniazid (MBC = 25 micrograms/ml).

Synthesis and evaluation of in vitro antimycobacterial activity of some 5-(5-nitro-2-thienyl)-2-(piperazinyl, piperidinyl and morpholinyl)-1,3,4-thiadiazole derivatives.

A new series of 5-(5-nitro-2-thienyl)-2-(piperazinyl, piperidinyl and morpholinyl)-1,3,4-thiadiazole derivatives(5a-g) have been synthesized and evaluated against Mycobacterium tuberculosis H37Rv as apart of TAACF TB screening program under direction of the US National Institute of Health, NIAID division. Primary screening was conducted at the single concentration, 6.25 mg/ml against Mycobacterium tuberculosis H37Rv (ATCC 27294) in BACTEC 12B medium using a broth microdilution assay, the Microplate Alamar Blue Assay (MABA). The minimum inhibitory concentration (MIC) determined for compounds demonstrating 90% growth inhibition in the primary screening. The tested compounds showed a varying degree of inhibitory activity (Inhibition = 0-100%). The most active compounds were 4-methyl and 4-benzoylpiperaxinyl analogues(5b and 5g) with the same MIC value of 3.13 micrograms/ml.

Diffusion-limited and advection-driven electrodeposition in a microfluidic channel.

Self-terminating electrochemical fabrication was used within a microfluidic channel to create a junction between two Au electrodes separated by a gap of 75 microm . During the electrochemical process of etching from the anode to deposition at the cathode, flow could be applied in the anode-to-cathode direction. Without applied flow, dendritic growth and dense branching morphologies were typically observed at the cathode. The addition of applied flow resulted in a densely packed gold structure that filled the channel. A computer simulation was developed to explore regimes where the diffusion, flow, and electric field between the electrodes individually dominated growth. The model provided good qualitative agreement relating flow to the experimental results. The model was also used to contrast the effects of open and closed boundaries and electric field strength, as factors related to tapering.

Source-sink partitioning. Do we need Münch?

The simulation of phloem translocation by the Münch theory commonly uses resistances from sources to sinks: the resistances are therefore regarded as important in partitioning. Although resistance is generally a set constant, it is in fact strongly affected by viscosity, and thus the concentration of the transported solute. In this paper, the model of partitioning proposed by Minchin et al. was first corrected for variations in viscosity. The model was further modified, with the source considered as an activity of solute production rather than as a compartment concentration. When so defined, the source cannot differ from the sum of sink activities, largely outdating the source- or sink-limitation concepts. The corrected model confined the effect of resistances on the partitioning to low source activities. In the example of wheat grain filling analysed, such activities would be so low that they would correspond only to pathological conditions. In that case, the use of resistances in modelling is therefore just a mathematical burden, not even easily quantifiable since they are related to anatomical traits that are difficult to access. Leaving out resistances, it becomes easy to calculate the sink activities directly from the source activities, using an intuitive, accessible parametrization. The conditions for such a simplification are discussed.