Nucleotide Polymorphism Associated with Ethambutol Resistance in Clinical Isolates of Mycobacterium tuberculosis

Ethambutol (EMB) is a first-line drug used for antitubercular therapy in combination with other drugs as recommended by World Health Organization DOTS/DOTS-Plus regimens. EMB is also effective in the treatment of opportunistic mycobacterial infections in patients with human immunodeficiency virus. The emb locus has been considered as a drug target for EMB, and substitutions of codon 306 in Mycobacterium tuberculosis gene embB have been shown to be the most frequent and predictive mutations for EMB resistance. The aim of the present study was to detect embB and embC gene mutations in EMB-resistant clinical isolates. A total of 23 isolates of M. tuberculosis from patients with pulmonary tuberculosis were included in the study. Drug sensitivity was tested by proportion method and E-test. All 23 isolates were EMB resistant. Primers to amplify the embB and embC gene were designed, and polymerase chain reaction products were subjected for sequence analysis. H37Rv standard laboratory strain was used as control. Nucleotide sequencing showed that 16 strains had a mutation in the embB gene. The most common mutation observed in the embB gene was at codon 306, followed by mutations at codons 299 and 378 in 4 and 2 isolates, respectively. Novel mutations have been reported at codons 239, 240, 247, 282, 311, 368, 397, 446, 469, and 471. Sequence analysis of the embC gene showed mutation in 8 isolates at codon 270. Novel mutations in embC have been reported at codons 251 and 254. The most common nucleotide polymorphism in our isolates was at codons 306 and 299 in the embB gene and at codon 270 in the embC gene. A mutation at codon 306 was usually associated with high-level ethambutol resistance.     

Effects of prenatal diclofenac sodium exposure on newborn testis: a histomorphometric study

Diclofenac sodium (DS) is used primarily to treat fever and to alleviate pain and inflammation. We investigated the effects of DS exposure during gestation on the testes of rat pups to investigate the safety of its use during the prenatal period. Pregnant rats were separated into control, saline, low dose, medium dose and high dose groups. DS was given between weeks 15 and 21 of gestation. Total numbers of spermatogonia and Sertoli cells were counted in the testes of 7-day-old male rats using the physical disector method. By the end of the study, the total number of Sertoli cells was decreased significantly in a dose dependent manner in the medium and high dose groups compared to controls. No significant differences were found in the total number of spermatogonia in the control, saline and low dose DS groups. Medium and high dose DS administration reduced the total number of spermatogonia compared to other groups. We suggest that prenatal administration of DS can cause deleterious effects on the testis development, especially in high doses.     

Farnesyl Phosphatase,a Corpora allata Enzyme Involved in Juvenile Hormone Biosynthesis in Aedes aegypti

Background

The juvenile hormones (JHs) are sesquiterpenoid compounds that play a central role in insect reproduction, development and behavior. The late steps of JH III biosynthesis in the mosquito Aedes aegypti involve the hydrolysis of farnesyl pyrophosphate (FPP) to farnesol (FOL), which is then successively oxidized to farnesal and farnesoic acid, methylated to form methyl farnesoate and finally transformed to JH III by a P450 epoxidase. The only recognized FPP phosphatase (FPPase) expressed in the corpora allata (CA) of an insect was recently described in Drosophila melanogaster (DmFPPase). In the present study we sought to molecularly and biochemically characterize the FPP phosphatase responsible for the transformation of FPP into FOL in the CA of A. aegypti.

Methods

A search for orthologs of the DmFPPase in Aedes aegypti led to the identification of 3 putative FPPase paralogs expressed in the CA of the mosquito (AaFPPases-1, -2, and -3). The activities of recombinant AaFPPases were tested against general phosphatase substrates and isoprenoid pyrophosphates. Using a newly developed assay utilizing fluorescent tags, we analyzed AaFPPase activities in CA of sugar and blood-fed females. Double-stranded RNA (dsRNA) was used to evaluate the effect of reduction of AaFPPase mRNAs on JH biosynthesis.

Conclusions

AaFPPase-1 and AaFPPase-2 are members of the NagD family of the Class IIA C2 cap-containing haloalkanoic acid dehalogenase (HAD) super family and efficiently hydrolyzed FPP into FOL. AaFPPase activities were different in CA of sugar and blood-fed females. Injection of dsRNAs resulted in a significant reduction of AaFPPase-1 and AaFPPase-2 mRNAs, but only reduction of AaFPPase-1 caused a significant decrease of JH biosynthesis. These results suggest that AaFPPase-1 is predominantly involved in the catalysis of FPP into FOL in the CA of A. aegypti.     

Fate of pathogenic and non-pathogenic Escherichia coli strains in two fermented milk products

F eresu , S. & N yati , H. 1990. Fate of pathogenic and non-pathogenic Escherichia coli strains in two fermented milk products. Journal of Applied Bacteriology 69 , 814–821.
The growth and survival of pathogenic and non-pathogenic strains of Escherichia coli was determined in traditionally fermented pasteurized and unpasteurized milk and in Lacto, an industrially fermented milk. Each milk treatment was incubated at 20C for 24 h and then stored at either 20C or 5C for 96 h.
Lacto inhibited all the three E. coli strains. Two strains could not be recovered and the third survived only in very low numbers after 24 h storage of Lacto at both 20C and 5C. All three E. coli strains survived and multiplied to maximum cell numbers in the range 10⁷-10⁹/ml during traditional fermentation of unpasteurized milk. Cell numbers decreased to 10³-10⁶ and 10²-10⁵ during storage of the fermented product at 20C and 5C respectively. Higher maximum numbers, 10⁹-10¹⁰, of the three strains of E. coli were attained during traditional fermentation of pasteurized milk. The numbers decreased to 10⁵-10⁸ and 10⁴-10⁷ during storage of the fermented product at 20C and 5C respectively. Generally, fewer E. coli survived when the fermented milk products were stored at refrigeration temperature.     

Patterns of Biofilm Succession on a Sheltered Rocky Shore in Hong Kong

Successional patterns are dependent on the nature of the substratum, water flow, concentrations of organics as well as the availability of bacteria, algal spores and invertebrate larvae in the coastal environment. Bacteria play an especially important role in biofilm formation as they are generally the earliest colonizers. In the present study, both winter and summer biofilm succession patterns were examined on glass coverslips inverted on experimental racks attached at two tidal levels on a sheltered shore in Hong Kong. In the succession, bacteria were followed by diatoms and cyanobacteria. Encrusting algae appeared in the late stages of the experiment (day 80 in summer and day 60 in winter). Colonization by bacteria was much slower in summer and their density remained low throughout the experimental period. The first appearance of diatoms and cyanobacteria, however, was more rapid in the summer. Bacteria and diatoms on the low-shore surfaces also had a faster succession rate than on the high-shore surfaces, suggesting that desiccation/aerial temperature are the causal factors for such differences.     

High throughput screening to investigate the interaction of stem cells with their extracellular microenvironment

Stem cells in vivo are housed within a functional microenvironment termed the “stem cell niche.” As the niche components can modulate stem cell behaviors like proliferation, migration and differentiation, evaluating these components would be important to determine the most optimal platform for their maintenance or differentiation. In this review, we have discussed methods and technologies that have aided in the development of high throughput screening assays for stem cell research, including enabling technologies such as the well-established multiwell/microwell plates and robotic spotting, and emerging technologies like microfluidics, micro-contact printing and lithography. We also discuss the studies that utilized high throughput screening platform to investigate stem cell response to extracellular matrix, topography, biomaterials and stiffness gradients in the stem cell niche. The combination of the aforementioned techniques could lay the foundation for new perspectives in further development of high throughput technology and stem cell research.     

Health-related quality of life and functional ability in patients with early arthritis during remission steered treatment: results of the IMPROVED study

Introduction

The aim of this study was to investigate patient reported outcomes (PROs) of functional ability and health related quality of life (HRQoL) in patients with early (rheumatoid) arthritis during one year of remission steered treatment.

Methods

In this study, 610 patients with early rheumatoid arthritis (RA) or undifferentiated arthritis (UA) were treated with methotrexate (MTX) and tapered high dose of prednisone. Patients in early remission (Disease Activity Score (DAS) <1.6 after 4 months) tapered prednisone to zero and when in persistent remission, also tapered MTX. Patients not in early remission were randomized to either MTX + hydroxychloroquine + sulphasalazine + prednisone (arm 1) or to MTX + adalimumab (arm 2). Every 4 months, patients filled out the Health Assessment Questionnaire (HAQ) and the McMaster Toronto Arthritis Patient Preference Questionnaire (MACTAR), the Short Form 36 (SF-36) and visual analogue scales (VAS). Change scores were compared between treatment groups. The association with achieving remission was analyzed using linear mixed models.

Results

During year 1, patients who achieved early remission had the most improvement in PROs with scores comparable to the general population. Patients in the randomization arms showed less improvement. Scores were comparable between the arms. There was a significant association between achieving remission and scores of HAQ, MACTAR and physical HRQoL.

Conclusions

In early arthritis, PROs of functional ability and HRQoL after one year of remission steered treatment reach normal values in patients who achieved early remission. In patients not in early remission, who were randomized to two strategy arms, PROs improved less, with similar scores in both treatment arms.

Trial registrations

ISRCTN11916566 and EudraCT2006-006186-16     

Negative Feedbacks by Isoprenoids on a Mevalonate Kinase Expressed in the Corpora Allata of Mosquitoes

Background

Juvenile hormones (JH) regulate development and reproductive maturation in insects. JHs are synthesized through the mevalonate pathway (MVAP), an ancient metabolic pathway present in the three domains of life. Mevalonate kinase (MVK) is a key enzyme in the MVAP. MVK catalyzes the synthesis of phosphomevalonate (PM) by transferring the γ-phosphoryl group from ATP to the C₅ hydroxyl oxygen of mevalonic acid (MA). Despite the importance of MVKs, these enzymes have been poorly characterized in insects.

Results

We functionally characterized an Aedes aegypti MVK (AaMVK) expressed in the corpora allata (CA) of the mosquito. AaMVK displayed its activity in the presence of metal cofactors. Different nucleotides were used by AaMVK as phosphoryl donors. In the presence of Mg²⁺, the enzyme has higher affinity for MA than ATP. The activity of AaMVK was regulated by feedback inhibition from long-chain isoprenoids, such as geranyl diphosphate (GPP) and farnesyl diphosphate (FPP).

Conclusions

AaMVK exhibited efficient inhibition by GPP and FPP (Ki less than 1 μM), and none by isopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DPPM). These results suggest that GPP and FPP might act as physiological inhibitors in the synthesis of isoprenoids in the CA of mosquitoes. Changing MVK activity can alter the flux of precursors and therefore regulate juvenile hormone biosynthesis.     

Molecular imaging of akt enables early prediction of response to molecular targeted therapy

Development of noninvasive, real-time molecular imaging tools to assess responsiveness of a given therapy may be a critical component of the success of individualized therapy approach for patients. Toward this, we have previously developed and validated molecular sensors for Akt and caspase-3 activity, and in this report, we have explored the utility of these reporters in assessing the responsiveness of tumors to a combination of gemcitabine (Gem) and cetuximab (Cet) delivered in two opposite schedules. We found that human head and neck cancer (UMSCC1) xenografts responded significantly better in a schedule where cetuximab was administered after gemcitabine when compared with the schedule of cetuximab followed by gemcitabine. Wilcoxon two-sample tests suggested that the difference in tumor volumes in two schedules became significant on day 7 (P > .05 on day 4, and P < .05 on days 7 and 10), and the difference in activity of Akt in two schedules became significant on day 4 (P < .05 on days 4, 6, and 10). Using Akt reporter activity and cubic spline interpolation, the distinction between the two schedules could be detected 2 days before using the tumor volume, suggesting that molecular imaging of Akt may allow early prediction of therapy responsiveness. We did not observe a significant difference between the two schedules in the caspase-3 activity. In summary, this proof-of-concept study provides a basis for using molecular imaging of Akt as an early indicator of therapeutic efficacy.