Colistin A and colistin B among inhibitory substances of <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> JB05-01-1

Recently, we isolated and reported the antagonism of Paenibacillus polymyxa JB05-01-1 (P. polymyxa JB05-01-1) against Gram-negative bacteria. Here, we provide more insights and attribute the abovementioned antagonism to the production of colistins A and B, which were purified by Amberlite column exchange, C18 column hydrophobicity, superdex 75 16/60 gel filtration chromatography connected to fast protein liquid chromatography and identified by MALDI TOF/TOF, and manual nanospray analysis. The amount of colistin A and colistin B recovered from 500 ml of culture supernatant was about 0.05 mg. The specific activity and the average recovery of the eluted substances were 5,120 AU/mg and 1.1%, respectively. The minimal inhibitory concentrations of the purified colistins against Escherichia coli O157:H7 and Pseudomonas fluorescens LRC R73 were 0.13 and 0.62 μg/ml, respectively.     

Development and application of an in-house reverse hybridization method for Chlamydia trachomatis genotyping

Aim

To develop and evaluate an in‐house reverse hybridization technique for Chlamydia trachomatis genotype identification.

Methods and Results

The evaluation of the developed and optimized reverse hybridization method on reference strains showed the specific detection of all genotypes. This technique showed its ability to type one inclusion‐forming unit of C. trachomatis genotype E and equivalent sensitivity to the Cobas TaqMan assay. It was also able to detect mixed infections in vitro. Application of the reverse hybridization method on 38 isolated C. trachomatis strains and their respective swabs allowed the detection of six urogenital genotypes D, E, F, G, H and K and one trachoma genotype B. Genotype E was the most prevalent, detected in 73% of the swab samples. Mixed infections were detected in 26% of swab cases.

Conclusion

The reverse hybridization technique is simple and does not require specialized instruments. It is powerful in the diagnosis of mixed infections and is suitable for use in epidemiological studies.

Significance and Impact of the Study

This technique allowed rapid C. trachomatis genotype identification.     

<Emphasis Type="Italic">Bacillus subtilis</Emphasis> bacteriocin Bac 14B with a broad inhibitory spectrum: Purification,amino acid sequence analysis,and physicochemical characterization

Bacillus subtilis strain 14B was used to produce a novel antimicrobial peptide (bacteriocin) called Bac 14B. Pure bacteriocin was obtained after heat and acidic treatments (80°C and pH 4), precipitation by ammonium sulfate, and chromatography on Sephadex G-50 and Mono Q Sepharose columns. Based on MALDI-TOF mass spectrometry analysis, purified Bac 14B is a monomer protein with a molecular mass of 20110.13 Da. N-terminal sequencing allowed for the straightforward identification of its first 12 residues, which were of a pure bacteriocin. It also revealed that this bacteriocin contained a unique sequence, namely M-L-K-A-N-L-Q-N-P-L-N-A, suggesting the identification of a novel compound. Bac 14B was stable for 1 h at temperatures up to 80°C and pH of 4 ∼ 8. It also proved sensitive to various proteases, which demonstrated its protein nature. Bac 14B displayed a bacteriolytical mode of action and a broad range of inhibitory spectra toward Gram-positive and -negative pathogens. Interestingly, based on conventional agronomic seed vigor parameters, the application of Bac 14B (500 activity units/mL) to various crops revealed that this bacteriocin was a potent exogenous enhancer of growth that stimulated the seedling vigor of tomatoes and muskmelons. Compared to those of the control, the germination percentage, shoot weight, shoot height, and root length were all significantly enhanced in Bac 14B-treated plant seeds. Bac 14B also exhibited effective disinfectant properties against a wide range of seedborne diseases and significant effects on the control of damping off diseases, particularly at the pregermination stage. It also proved to be effective against root rot diseases caused by Alternaria solani and other bacterial seedborne pathogens such as wilt diseases. The findings indicate that Bac 14B is the first B. subtilis-produced bacteriocin ever reported to exhibit such promising biological properties.     

Actin depolymerizing factors ADF7 and ADF10 play distinct roles during pollen development and pollen tube growth

An important player in actin remodeling is the actin depolymerizing factor (ADF) which increases actin filament treadmilling rates. Previously, we had prepared fluorescent protein fusions of two Arabidopsis pollen specific ADFs, ADF7 and ADF10. These had enabled us to determine the temporal expression patterns and subcellular localization of these proteins during male gametophyte development. Here we generated stable transformants containing both chimeric genes allowing for simultaneous imaging and direct comparison. One of the striking differences between the two proteins was the localization profile in the growing pollen tube apex. Whereas ADF10 was associated with the filamentous actin array forming the subapical actin fringe, ADF7 was present in the same cytoplasmic region, but in diffuse form. This suggests that ADF7 is involved in the high actin turnover that is likely to occur in the fringe by continuously and efficiently depolymerizing filamentous actin and supplying monomeric actin to the advancing end of the fringe. The possibility to visualize both of these pollen-specific ADFs simultaneously opens avenues for future research into the regulatory function of actin binding proteins in pollen.     

The effect of apolipoprotein E polymorphism on plasma cholesteryl ester transfer protein activity in type 2 diabetic patients

We studied the relationship between apo E polymorphism and cholesteryl ester transfer protein (CETP) activity in 127 type 2 diabetic patients who did not take lipid lowering drugs. Furthermore, we studied the relationship between apo E and cholesteryl ester transfer protein (CETP) in modulating plasma triglyceride and HDLcholesterol. Apo E genotypes were determined by PCR-RFLP, and CETP activity was measured using an exogenous way. Our results showed that the CETP activity increased significantly in the E2 carrier group compared to E4 carriers and E3/E3 homozygous (84.7 ± 43.9 vs. 62.5 ± 35.9 vs. 52.6 ± 23.6 nmol CE/ml/2h, respectively; p = 0.015). However, there was no association between apo E polymorphism and lipid parameter variations. Even after adjustment for CETP activity, the results remained unchanged, showing that CETP did not step in the relationship between apo E and lipid parameter variations. In conclusion there is an association between apo E polymorphism and CETP activity, and this association did not affect the relationship between apo E polymorphism and triglyceride and HDLcholesterol concentrations. Published in Russian in Molekulyarnaya Biologiya, 2008, Vol. 42, No. 6, pp. 931–935. The text was submitted by the authors in English.     

Nutritional regulation and kinetics of flocculosin synthesis by Pseudozyma flocculosa

This study sought to identify the factors and conditions that affected production of the antifungal glycolipid flocculosin by the biocontrol agent Pseudozyma flocculosa. For this purpose, different parameters known or reported to influence glycolipid release in fungi were tested. Concentration of the start-up inoculum was found to play an important role in flocculosin production, as the optimal level increased productivity by as much as tenfold. Carbon availability and nitrogen source (i.e., organic vs inorganic) both had a direct influence on the metabolism of P. flocculosa, leading to flocculosin synthesis. In general, if conditions were conducive for production of the glycolipid, carbon availability appeared to be the only limiting factor. On the other hand, if yeast extract was supplied as nitrogen source, fungal biomass was immediately stimulated to the detriment of flocculosin synthesis. Unlike other reports of glycolipid release by yeast-like fungi, inorganic nitrogen starvation did not trigger production of flocculosin. The relationship between the factors influencing flocculosin production in vitro and the conditions affecting the release of the molecule by P. flocculosa in its natural habitat appears to be linked to the availability of a suitable and plentiful food source for the biocontrol agent.     

Improving performance of olive trees by the enhancement of key physiological parameters of olive leaves in response to foliar fertilization

Most of the studies investigated the effects of nutrient-based fertilizers on olive fruits and oil quality; few studies have been interested in the modification of the chemical composition of olive leaves in response to fertilization. Thus, the current study aims to examine the effects of foliar fertilization on the mineral profile of olive leaves as well as the concentrations of chlorophyll, antioxidants (phenolic compounds) and carbohydrates. Experimentation consists of the annual application of six treatments during two successive growing seasons (2009–2010): TC (untreated trees), P1 (nitrogen-based fertilizer), P2 (contains boron, magnesium and manganese), P3 (phosphorus and potassium based fertilizer), P4 (rich in calcium and phosphorus), T5 (P1 and P2 application) and T6 (P1, P2, P3 and P4 application). At the end of the experiment, mineral analysis of olive leaves showed an increase in the concentrations of most nutrients which induced changes in biochemical composition: an increase of chlorophyll content, a reduction of total phenols and oleuropein concentrations coupled with an increase of hydroxytyrosol level. Moreover, an increase of total sugar content and most individual sugars, principally translocated forms of sugars (mannitol, sucrose and raffinose), was also observed. The accumulation of these key physiological parameters by foliar fertilization suggests an improvement of physiological performance and photosynthetic capacity of olive trees. Moreover, from a biological point of view, the results of the study revealed the possibility to improve plants of medicinal interest by enhancing the accumulation of some bio-active compounds, such as hydroxytyrosol and mannitol, via foliar nutrient supply.     

The Management of Chronic Pulmonary Aspergillosis: The UK National Aspergillosis Centre Approach

Purpose of Review

Chronic pulmonary aspergillosis (CPA) is a serious long-term fungal disease of the lung with a worldwide prevalence. Treatment of CPA is not straightforward given the often-multiple associated co-morbidities, complex clinical picture, drug interactions, toxicities and intolerances.

Recent Findings

First line treatment is oral itraconazole or voriconazole. In the event of intolerance or toxicity, patients may be swapped from itraconazole to voriconazole or vice versa. In the event of resistance or further intolerance, third line treatment with posaconazole could be initiated. In those with pan-azole resistance, short-term courses of intravenous liposomal amphotericin B or micafungin are fourth line therapy, keeping in mind the nephrotoxic effects of amphotericin B.

Summary

The available evidence for current treatments in CPA is limited and based mostly on retrospective cohort studies. There is a real need to raise awareness of this devastating disease to enable early treatment as well as prospective drug trials and studies to identify potential patient factors that correlate with progression, severity and overall outcomes in order to target future therapies.

     

Evaluation of an <Emphasis Type="Italic">in silico</Emphasis> predicted specific and immunogenic antigen from the OmcB protein for the serodiagnosis of <Emphasis Type="Italic">Chlamydia trachomatis</Emphasis> infections

Background  

The OmcB protein is one of the most immunogenic proteins in C. trachomatis and C. pneumoniae infections. This protein is highly conserved leading to serum cross reactivity between the various chlamydial species. Since previous studies based on recombinant proteins failed to identify a species specific immune response against the OmcB protein, this study evaluated an in silico predicted specific and immunogenic antigen from the OmcB protein for the serodiagnosis of C. trachomatis infections.